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The potent immunosuppression induced by glioblastoma (GBM) is one of the primary obstacles to finding effective immunotherapies. One hallmark of the GBM-associated immunosuppressive landscape is the massive infiltration of myeloid-derived suppressor cells (MDSC) and, to a lesser extent, regulatory T cells (Treg) within the tumor microenvironment. Here, we showed that regulatory B cells (Breg) are a prominent feature of the GBM microenvironment in both preclinical models and clinical samples. Forty percent of GBM patients (n = 60) scored positive for B-cell tumor infiltration. Human and mouse GBM-associated Bregs were characterized by immunosuppressive activity toward activated CD8+ T cells, the overexpression of inhibitory molecules PD-L1 and CD155, and production of immunosuppressive cytokines TGFβ and IL10. Local delivery of B cell–depleting anti-CD20 immunotherapy improved overall survival of animals (IgG vs. anti-CD20 mean survival: 18.5 vs. 33 days, P = 0.0001), suggesting a potential role of Bregs in GBM progression. We unveiled that GBM-associated MDSCs promoted regulatory B-cell function by delivering microvesicles transporting membrane-bound PD-L1, able to be up-taken by tumoral B cells. The transfer of functional PD-L1 via microvesicles conferred Bregs the potential to suppress CD8+ T-cell activation and acquisition of an effector phenotype. This work uncovered the role of B cells in GBM physiopathology and provides a mechanism by which the GBM microenvironment controls B cell–mediated immunosuppression.See related Spotlight on p. 1902

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Immunotherapy has revolutionized cancer treatment but has yet to be translated into brain tumors. Studies in other solid tumors suggest a central role of B-cell immunity in driving immune-checkpoint-blockade efficacy. Using single-cell and single-nuclei transcriptomics of human glioblastoma and melanoma brain metastasis, we found that tumor-associated B-cells have high expression of checkpoint molecules, known to block B-cell-receptor downstream effector function such as plasmablast differentiation and antigen-presentation. We also identified TGFβ-1/TGFβ receptor-2 interaction as a crucial modulator of B-cell suppression. Treatment of glioblastoma patients with pembrolizumab induced expression of B-cell checkpoint molecules and TGFβ-receptor-2. Abrogation of TGFβ using different conditional knockouts expanded germinal-center-like intratumoral B-cells, enhancing immune-checkpoint-blockade efficacy. Finally, blocking αVβ8 integrin (which controls the release of active TGFβ) and PD-1 significantly increased B-cell-dependent animal survival and immunological memory. Our study highlights the importance of intratumoral B-cell immunity and a remodeled approach to boost the effects of immunotherapy against brain tumors.

Sf6 is a bacterial virus that infects the human pathogen Shigella flexneri. Here, we describe the cryo–electron microscopy structure of the Sf6 tail machine before DNA ejection, which we determined at a 2.7-angstrom resolution. We built de novo structures of all tail components and resolved four symmetry-mismatched interfaces. Unexpectedly, we found that the tail exists in two conformations, rotated by ~6° with respect to the capsid. The two tail conformers are identical in structure but differ solely in how the portal and head-to-tail adaptor carboxyl termini bond with the capsid at the fivefold vertex, similar to a diamond held over a five-pronged ring in two nonidentical states. Thus, in the mature Sf6 tail, the portal structure does not morph locally to accommodate the symmetry mismatch but exists in two energetic minima rotated by a discrete angle. We propose that the design principles of the Sf6 tail are conserved across P22-like Podoviridae.

Aims The aim of this study was to analyse the necessity of routine group and saves (G+S) in patients undergoing laparoscopic cholecystectomies and to establish whether it would be safe to cease this practice. In doing so, we aim to reduce financial burden and workload, whilst maintaining safe clinical outcomes. Methods This was a retrospective study into all consecutive patients that underwent an elective or emergency laparoscopic cholecystectomy in Royal Hampshire County Hospital (RHCH) from May 2019 - May 2021. IT systems and clinical notes were analysed to identify the quantity of pre-operative G+S samples sent, and the proportion of patients that required a postoperative blood transfusion. Results Over 24 months, 427 laparoscopic cholecystectomies were performed in RHCH, 70% of which were elective. In total, 682 G+S samples were processed (1.6/patient), resulting in a cost of £16504. Two patients received a postoperative blood transfusion (0.5%). One of these patients presented with an UGI bleed, whilst the other presented with a haemolytic crisis. They were therefore predictable, pre-operative conditions, for which a post-operative transfusion had been planned prior to surgery. Conclusion In conclusion, this study suggests that performing routine G+S for all laparoscopic cholecystectomy patients has created an unnecessary workload and financial burden on this small DGH. Furthermore, during this 24 months, this practice had no clinical advantage. We therefore advise that sending pre-operative G+S samples should be a clinical decision made on a case by case basis, rather than a default for all patients.

Pseudomonas phages are increasingly important biomedicines for phage therapy, but little is known about how these viruses package DNA. This paper explores the terminase subunits from the Myoviridae E217, a Pseudomonas-phage used in an experimental cocktail to eradicate P. aeruginosa in vitro and in animal models. We identified the large (TerL) and small (TerS) terminase subunits in two genes ~58 kbs away from each other in the E217 genome. TerL presents a classical two-domain architecture, consisting of an N-terminal ATPase and C-terminal nuclease domain arranged into a bean-shaped tertiary structure. A 2.05 Å crystal structure of the C-terminal domain revealed an RNase H-like fold with two magnesium ions in the nuclease active site. Mutations in TerL residues involved in magnesium coordination had a dominant-negative effect on phage growth. However, the two ions identified in the active site were too far from each other to promote two-metal-ion catalysis, suggesting a conformational change is required for nuclease activity. We also determined a 3.38 Å cryo-EM reconstruction of E217 TerS that revealed a ring-like decamer, departing from the most common nonameric quaternary structure observed thus far. E217 TerS contains both N-terminal helix-turn-helix motifs enriched in basic residues and a central channel lined with basic residues large enough to accommodate double-stranded DNA. Overexpression of TerS caused a more than a four-fold reduction of E217 burst size, suggesting a catalytic amount of the protein is required for packaging. Together, these data expand the molecular repertoire of viral terminase subunits to Pseudomonas-phages used for phage therapy.

Glioblastoma (GBM) continues to retain its dismal prognosis despite numerous new therapeutic modalities to target various aspects of the tumor. There is a need to identify new, and even personalized, targetable GBM specific antigens. Our B-cell-based vaccine (BVax) is generated by isolating immune experienced B cells, identified by 4-1BBL, from murine secondary lymphoid organs or patient blood. We have shown this subset of B cells to have anti-tumoral potential in GBM. These cells are strengthened with BAFF, CD40, and IFNg stimulation to form BVax and then activated in vitro to form plasmablasts. Immunoprecipitation-mass spectrometry is performed using BVax-derived antibodies and tumor lysate from the paired patient specimen. We have identified unique antigens bound by BVax-derived antibodies. Given the significant therapeutic benefit we have seen using BVax-derived antibodies in preclinical murine models, we hypothesize that targeting these unique antigens would have significant therapeutic benefit in GBM patients. We focused on antigens that showed a survival benefit with lower expression using CGGA database analysis. We then confirm the presence and prevalence of the antigens within tumor cells in the patient’s tissue using immunohistochemistry, identifying some antigens in up to 15% of tumor cells. Using our brain tumor bank repository, we screened other GBM patient’s tissue for the presence of these markers to determine the broader applicability of targeting each antigen. We have identified proteins key in extracellular matrix formation which promote tumor growth and progression in cancer, including the ability to induce epithelial-mesenchymal transition. Simultaneously we have developed a computational algorithm to predict which antigens BVax-derived antibodies will bind using single cell RNA sequencing data from GBM patient-derived tumor and BVax, which may have broader applicability to a larger number of patients. Both techniques serve as exciting platforms to identify new therapeutic targets in GBM.

Objective: Aortic endothelial cell dysfunction is an early trigger of atherosclerosis, the major cause of cardiovascular disease. Nanomedicines targeting vascular endothelium and lesions hold great promise as therapeutic solutions to vascular disorders. The objective of the present study is to investigate macrovascular targeting efficacy of a redox-responsive degradable polyurethane-polyurea nanocapsule (Puua-NC). Approach and Results: Red fluorescent lipophilic cationic indocarbocyanine dye, with DiI as cargo, was encapsulated into Puua-NCs of variable sizes (20, 175, 255 nm in diameter) at a concentration of 8.60, 6.54, and 6.91ng/mg NCs, respectively. In vitro cellular uptake studies with human aortic endothelial cells (HAECs) indicated these different sized Puua-NCs were efficiently taken up by cells, and the cellular uptake of NCs was positively correlated with concentration and incubation time. Exposure of HAECs to dithiothreitol (DTT, a thiol reducing reagent) or diamide (a thiol oxidizing reagent) altered intracellular thiol redox status and broke disulfide bonds on the Puua-NC shell, triggering cell disassembly and release of the encapsulated fluorescent cargo DiI, as evidenced by the loss of bright punctuate red fluorescence and appearance of the weak diffused signals of DiI. Using ApoE deficient mice fed a Western-type diet, a mouse model of atherosclerosis, we showed that intravenously administrated Puua-NCs were efficiently taken up by aortic endothelium and atherosclerotic lesions in vivo . Importantly, internalized Puua-NCs in vascular cells and lesions were rapidly disassembled to release DiI in response to DTT treatment. Conclusions: Puua-NCs demonstrate remarkable affinity to aortic endothelium and atherosclerotic lesions, with disassembly well controlled by redox status. Puua-NCs may be used as a novel redox-responsive degradable nanodevice to deliver drugs and gene materials to the cardiovascular system.

Portal proteins are dodecameric assemblies that occupy a unique 5-fold vertex of the icosahedral capsid of tailed bacteriophages and herpesviruses. The portal vertex interrupts the icosahedral symmetry, and in vivo, its assembly and incorporation in procapsid are controlled by the scaffolding protein. Ectopically expressed portal oligomers are polymorphic in solution, and portal rings built by a different number of subunits have been documented in the literature. In this paper, we describe the cryo-EM structure of the portal protein from the Pseudomonas-phage PaP3, which we determined at 3.4 Å resolution. Structural analysis revealed a dodecamer with helical rather than rotational symmetry, which we hypothesize is kinetically trapped. The helical assembly was stabilized by local mispairing of portal subunits caused by the slippage of crown and barrel helices that move like a lever with respect to the portal body. Removing the C-terminal barrel promoted assembly of undecameric and dodecameric rings with quasi-rotational symmetry, suggesting that the barrel contributes to subunits mispairing. However, ΔC-portal rings were intrinsically asymmetric, with most particles having one open portal subunit interface. Together, these data expand the structural repertoire of viral portal proteins to Pseudomonas-phages and shed light on the unexpected plasticity of the portal protein quaternary structure.

While immunotherapy is used clinically to treat many cancers, its translation into brain tumors remains elusive. The importance of B cells in cancer immunity has become increasingly clear, and we previously developed a B cell-based cellular vaccine (BVax) against glioblastoma (GBM) by further activating 4-1BBL+ B cells with CD40 agonism and IFNγ. BVax were characterized as professional antigen-presenting cells (APCs) that promote CD8+ T cell migration and persistence in murine tumor-bearing brains. This study seeks to understand the mechanisms underlying BVax-induced CD8+ T cell fitness in the tumor microenvironment. Initial transcriptomic analysis highlighted that Bvax express high levels of IL15Rα, indicating their potential ability to trans-present IL15. Considering IL15 trans-presentation is fundamental in T-cell memory differentiation, we used BVax to induce T cell activation in the presence of exogenous IL15. BVax were better capable of activating antigen-specific CD8+ T cells and promoting a memory phenotype when compared to other professional APCs such as dendritic cells (DCs). T cell receptor (TCR) CDR3β sequencing showed that BVax expanded a number of TCR clones in-vitro that were found in brains of CT2A tumor-bearing mice in-vivo. These BVax-activated CD8+ T cells displayed a stronger antigen recall response and unique metabolic profile compared to DC-activated CD8+ T cells as shown by metabolomic analysis of tumor-infiltrating CD8+ T cells. When comparing the anti-tumor effects of CD8+ T cells activated by various APCs, BVax with exogenous IL15 promoted CD8+ T cells that displayed the most potent cytotoxicity against GBM cells in-vitro. Collectively, this study suggests that the IL15/IL15Rα axis and interactions with CD8+ T cell are key factors of BVax therapy in promoting a robust survival benefit and long-term immunologic memory against GBM in preclinical models. Additionally, the development of T cell therapies based on B cell licensing can be a promising future approach for glioblastoma therapy.

BACKGROUND: T cell receptor (TCR)-based cell therapy to treat solid tumors by targeting tumor neoantigens is gaining momentum. While there is an urgent need for discovering novel TCRs, one challenge is that the exogenous TCR may compete with the endogenous TCR for CD3 signaling components and form mixed dimers with potential toxicities. Additionally, the activity of T cells highly depends on signaling machinery only present in T cells, which restricts the scope of eligible therapeutic cells. We developed a TCR-like chimeric antigen-receptor to both overcome the mispairing of TCR α/β chains and to expand the scope of TCR-based cell therapy to other immune cells. METHOD: To identify novel TCRs, a monoclonal TCR discovery platform was developed. A high avidity TCR of a neoepitope-specific CD8+ T cell clone (CTL) that was generated from donor PBMCs was isolated and sequenced. A chimeric antigen-receptor comprising an antigen binding domain (which is a portion of the identified TCR), a CD28 segment containing the hinge/transmembrane domain, and a cytoplasmic signaling domain containing a Dap10 and a Dap12 intracellular domains. To augment the avidity of the antigen receptor, a chimeric co-receptor was constructed by combining HLA-I binding domains of CD8α with the hinge/transmembrane/cytoplastic domains of CD4. T cell line (J.RT3-T3.5) and NK cell line (KYGH-1) were transduced by a lentivirus encoding the chimeric antigen-receptor and co-receptor to assess their expression and function. RESULTS: Two novel TCRs specific for CTNNB1S37F peptide (YLDSGIHFGA) and for p53R175H peptide (HMTEVVRHC) respectively were identified; both are high-avidity and can recognize the endogenous neoepitope presented by HLA-A2 in tumor cells. A chimeric antigen-receptor specific for CTNNB1S37F neoepitope/HLA-A2 was expressed on the surface of 293T cells independent of endogenous CD3. However, its avidity to recognize CTNNB1S37F neoepitope was approximately 5 folds lower than that of the native TCR. Co-expression of the co-receptor could significantly enhance the avidity of the chimeric antigen-receptor and make it capable of recognizing endogenously presented neoepitope in tumor cells. In addition, NK cells re-directed with the TCR-like chimeric antigen-receptor could also recognize the neoepitope peptide in the context of HLA-A2. CONCLUSION: We validated a TCR-like chimeric antigen-receptor that can be expressed on cells independent of endogenous CD3 and can recognize neoepitope/HLA-A2. We also designed and tested a co-receptor that can enhance the avidity of the chimeric antigen-receptor. To further leverage the avidity of the TCR-like chimeric antigen-receptor, testing of new configurations of the chimeric antigen-receptor and co-receptor is underway. Citation Format: Yafei Hou, David Hou. T cell receptor-like chimeric antigen-receptor to recognize neoepitopes derived from driver mutations [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B32.

Immunotherapy has revolutionized cancer treatment but has yet to be translated into brain tumors. Studies in melanoma and sarcoma, amongst other models, have revealed the accumulation of germinal-center-like B cells as a key survival predictor post-PD1 blockade. We seek to leverage B cell immunity to enhance immunotherapy effectiveness in glioblastoma (GBM). In human GBM and murine glioma models, we found that B cells in the tumor microenvironment (TME) are activated, but the expression of co-inhibitory molecules such as CD32 and CD72 blocks downstream effector function. Transcriptomic analysis showed high expression of inhibitory TGFβ receptors on B cells and high levels of TGFβ1 cytokine in the TME. We showed direct inhibition of B cell function through TGFβ signaling that could be prevented with TGFβ receptor blockade. Spatial multiplex immunofluorescence analysis of the TME revealed that tumor and myeloid cells express high levels of TGFβ and are also near B cells, allowing for TGFβ-mediated B cell inhibition. Blocking the TGFβ pathway via transgenic mice with TGFβ receptor knockout on B cells or TGFβ cytokine knockouts in myeloid cells, or generation of a CT2A tumor line with TGFβ cytokine knockdown, all demonstrated a survival benefit and more germinal-center-like B cells. There was also increased T cell proliferation and anti-tumor cytotoxicity. Finally, inhibiting αVβ8 integrin, a required factor that releases active TGFβ, is a translatable approach that also increased B cell proliferation and animal survival. Dual treatment with αVβ8+PD1 blockade showed the most potent survival as well as immunological memory against tumor re-challenge. Analysis of the B and T cell compartments after dual treatment showed synergy, with robust cellular proliferation and functional differentiation of plasmablasts and effector T cells. Collectively, our study highlights the importance of B cells in the TME and a remodeled approach to boost the effects of immunotherapy against GBM.

The genome packaging motor of bacteriophages and herpesviruses is built by two terminase subunits, known as large (TerL) and small (TerS), both essential for viral genome packaging. TerL structure, composition, and assembly to an empty capsid, as well as the mechanisms of ATP-dependent DNA packaging, have been studied in depth, shedding light on the chemo-mechanical coupling between ATP hydrolysis and DNA translocation. Instead, significantly less is known about the small terminase subunit, TerS, which is dispensable or even inhibitory in vitro, but essential in vivo. By taking advantage of the recent revolution in cryo-electron microscopy (cryo-EM) and building upon a wealth of crystallographic structures of phage TerSs, in this review, we take an inventory of known TerSs studied to date. Our analysis suggests that TerS evolved and diversified into a flexible molecular framework that can conserve biological function with minimal sequence and quaternary structure conservation to fit different packaging strategies and environmental conditions.

The potent immunosuppression induced by glioblastoma (GBM) is one of the primary obstacles to finding effective immunotherapies. One hallmark of the GBM-associated immunosuppressive landscape is the massive infiltration of myeloid-derived suppressor cells (MDSC) and, to a lesser extent, regulatory T cells (Treg) within the tumor microenvironment. Here, we showed that regulatory B cells (Breg) are a prominent feature of the GBM microenvironment in both preclinical models and clinical samples. Forty percent of GBM patients (n = 60) scored positive for B-cell tumor infiltration. Human and mouse GBM-associated Bregs were characterized by immunosuppressive activity toward activated CD8+ T cells, the overexpression of inhibitory molecules PD-L1 and CD155, and production of immunosuppressive cytokines TGFβ and IL10. Local delivery of B cell–depleting anti-CD20 immunotherapy improved overall survival of animals (IgG vs. anti-CD20 mean survival: 18.5 vs. 33 days, P = 0.0001), suggesting a potential role of Bregs in GBM progression. We unveiled that GBM-associated MDSCs promoted regulatory B-cell function by delivering microvesicles transporting membrane-bound PD-L1, able to be up-taken by tumoral B cells. The transfer of functional PD-L1 via microvesicles conferred Bregs the potential to suppress CD8+ T-cell activation and acquisition of an effector phenotype. This work uncovered the role of B cells in GBM physiopathology and provides a mechanism by which the GBM microenvironment controls B cell–mediated immunosuppression. See related Spotlight on p. 1902

Herein, we introduce the efficient synthesis of Q-proline (Q-Pro) based, metal-binding macrocycles (QPM), which can display up to nine functional groups. Synthesis of eight QPM was achieved through standard Fmoc-SPPS and peptoid chemistry. QPM are disordered in the absence of a metal cation, as evidenced by NMR and a crystal structure of QPM-3 obtained through racemic crystallization. Addition of metal cations cause these macrocycles to adopt ordered, uniform core structures regardless of the functional groups. Alkylation of QPM allows for addition of reactive functional groups as the final step in a synthesis. Interestingly, the addition of secondary functional groups to the hydantoin amide position (R2) converts the proline ring from Cg-endo to Cg-exo, due to steric interactions.

Glioma-associated myeloid cells generate polyamines to survive and function within the acidic tumor microenvironment., Glioblastoma is characterized by the robust infiltration of immunosuppressive tumor-associated myeloid cells (TAMCs). It is not fully understood how TAMCs survive in the acidic tumor microenvironment to cause immunosuppression in glioblastoma. Metabolic and RNA-seq analysis of TAMCs revealed that the arginine-ornithine-polyamine axis is up-regulated in glioblastoma TAMCs but not in tumor-infiltrating CD8+ T cells. Active de novo synthesis of highly basic polyamines within TAMCs efficiently buffered low intracellular pH to support the survival of these immunosuppressive cells in the harsh acidic environment of solid tumors. Administration of difluoromethylornithine (DFMO), a clinically approved inhibitor of polyamine generation, enhanced animal survival in immunocompetent mice by causing a tumor-specific reduction of polyamines and decreased intracellular pH in TAMCs. DFMO combination with immunotherapy or radiotherapy further enhanced animal survival. These findings indicate that polyamines are used by glioblastoma TAMCs to maintain normal intracellular pH and cell survival and thus promote immunosuppression during tumor evolution.

Herein, we introduce the efficient synthesis of Q-proline (Q-Pro) based, metal-binding macrocycles (QPM), which can display up to nine functional groups. Synthesis of eight QPM was achieved through standard Fmoc-SPPS and peptoid chemistry. QPM are disordered in the absence of a metal cation, as evidenced by NMR and a crystal structure of QPM-3 obtained through racemic crystallization. Addition of metal cations cause these macrocycles to adopt ordered, uniform core structures regardless of the functional groups. Alkylation of QPM allows for addition of reactive functional groups as the final step in a synthesis. Interestingly, the addition of secondary functional groups to the hydantoin amide position (R2) converts the proline ring from Cg-endo to Cg-exo, due to steric interactions.

Summary Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome delivery in prokaryotes remains limited, especially for short-tailed phages of the Podoviridae family. These viruses expel mysterious ejection proteins found inside the capsid to form a DNA-ejectosome for genome delivery into bacteria. Here, we reconstitute the phage T7 DNA-ejectosome components gp14, gp15, and gp16 and solve the periplasmic tunnel structure at 2.7 A resolution. We find that gp14 forms an outer membrane pore, gp15 assembles into a 210 A hexameric DNA tube spanning the host periplasm, and gp16 extends into the host cytoplasm forming a ∼4,200 residue hub. Gp16 promotes gp15 oligomerization, coordinating peptidoglycan hydrolysis, DNA binding, and lipid insertion. The reconstituted gp15:gp16 complex lacks channel-forming activity, suggesting that the pore for DNA passage forms only transiently during genome ejection.

Immunotherapy has revolutionized the treatment of many tumors. However, most glioblastoma (GBM) patients have not, so far, benefited from such successes. With the goal of exploring ways to boost anti-GBM immunity, we developed a B-cell-based vaccine (BVax) that consists of 4-1BBL+ B cells activated with CD40 agonism and IFNg stimulation. BVaxmigrate to key secondary lymphoid organs and are proficient at antigen cross-presentation, which promotes both the survival and functionality of CD8+ T cells. A combination of radiation, BVax, and PD-L1 blockade conferred tumor eradication in 80% of treated tumor-bearing animals. This treatment elicited immunologic memory that prevented the growth of new tumors upon subsequent re-injection in cured mice. GBM patient-derived BVax were successful in activating autologous CD8+ T cells; these T cells showed a strong ability to kill autologous glioma cells. In addition to the role in activating CD8+ T cells, BVax produce tumor-specific antibodies able to control tumor growth via antibody-mediated cell cytotoxicity. In conclusion, BVax tackles GBM immunosurveillance escape by using both cellular (CD8+ T-cell activation) and humoral (anti-tumor antibody production) immunity. Our study provides an efficient alternative to current immunotherapeutic approaches that can be readily translated to the clinic.

Background Despite the tremendous effort in basic, translational and clinical research, the standard-of-care of patients with glioblastoma (GBM) has been virtually unchanged for the past two decades, aside from tumor-treating fields. GBM is one of the immunologically ‘coldest tumors’ where T-cell exclusion is at its maximum, and myeloid infiltration predominates. This is due to profound immunosuppression, the metabolically hostile microenvironment, and the low mutational burden of these tumors. Together, these barriers have hindered the development of effective immunotherapies. With the goal of exploring ways to boost anti-GBM immunity, we developed a B-cell-based vaccine (BVax) that consists of 4-1BBL+ B cells activated with CD40 agonism and IFNgamma stimulation. Methods Studies on B-cell-driven inflammation have identified a subset of B cells expressing the co-stimulatory marker 4-1BBL (or CD137L) capable of enhancing CD8+ T-cell anti-tumor cytotoxicity. Such activation was achieved through multiple mechanisms, including antigen presentation, T-cell co-stimulation (4-1BBL and CD86), and cytokine production (TNFalpha). Thus, 4-1BBL+ B cells could be utilized to boost anti-tumor CD8+ T-cell response. In order to stabilize their antigen presentation function in-vivo and avoid potential immunosuppressive functions, we activated 4-1BBL+ B cells using CD40 and IFNgamma receptor (IFNgR) ligation (designated as BVax, figure 1A), both of which were effective to enhance B-cell-mediated antigen presentation (figure 1B-E). In the present study, we explored the ability of BVax to inhibit GBM growth by promoting tumor-specific CD8+ T-cell immunity and production of tumor-reactive antibodies. BVax’s therapeutic effectiveness was examined both alone and in combination with radiation and checkpoint blockade. Results BVax migrate to key secondary lymphoid organs and are proficient at antigen cross-presentation (figure 2A), which promotes both the survival and functionality of CD8+ T cells (figure 2B and C). A combination of radiation, BVax, and PD-L1 blockade conferred tumor eradication in 80% of treated tumor-bearing animals (figure 2D and E). This treatment elicited immunologic memory that prevented the growth of new tumors upon subsequent re-injection in cured mice (figure 2D and E). GBM patient-derived BVax were successful in activating autologous CD8+ T cells; these T cells showed a strong ability to kill autologous glioma cells. In addition to the role in activating CD8+ T cells, BVax produce mainly IgGs able to react to tumor cells (figure 4A) and tumor-associated antigens (figure 4B). Treatment of glioma bearing-mice with BVax-derived IgG elicited extended animal survival (figure 4C). Statistical analysis: Data are shown as mean±SD for a continuous variable and number (percentages or optical density measures) for a categorical variable. Differences between two groups were analyzed by Student’s t-test. Differences among multiple groups were evaluated using one-way ANOVA with post hoc Tukey’s test followed by post hoc Dunn’s multiple tests as appropriate. Survival curves were generated via the Kaplan-Meier method and compared by log-rank test and multiple comparisons were adjusted using the Bonferroni method. All the tests are two-sided and p-values or Benjamini-Hochberg adjusted false discovery rates less than 0.05 were considered as significant. Statistical analyses were performed using SAS9.4 and GraphPad Prism7.03. Conclusions In conclusion, BVax tackles GBM immunosurveillance escape by using both cellular (CD8+ T-cell activation) and humoral (anti-tumor antibody production) immunity. Our study provides an efficient alternative to current immunotherapeutic approaches that can be readily translated to the clinic. Ethics Approval All human samples (tumor, peripheral blood, and frozen tissue) were collected by the Nervous System Tumor Bank at Northwestern University (NSTB) under the institutional review board (IRB) protocol N° STU00202003. All animal experimentation protocols are approved by the Institutional Animal Care and Use Committee (IACUC) under protocol # IS00002459 at Northwestern University. Acknowledgements In conclusion, BVax tackles GBM immunosurveillance escape by using both cellular (CD8+ T-cell activation) and humoral (anti-tumor antibody production) immunity. Our study provides an efficient alternative to current immunotherapeutic approaches that can be readily translated to the clinic.

Background: Pulmonary arterial hypertension (PAH) is a fatal disease characterized by profound vascular remodeling in which pulmonary arteries narrow because of medial thickening and occlusion by neointimal lesions, resulting in elevated pulmonary vascular resistance and right heart failure. Therapies targeting the neointima would represent a significant advance in PAH treatment; however, our understanding of the cellular events driving neointima formation, and the molecular pathways that control them, remains limited. Methods: We comprehensively map the stepwise remodeling of pulmonary arteries in a robust, chronic inflammatory mouse model of pulmonary hypertension. This model demonstrates pathological features of the human disease, including increased right ventricular pressures, medial thickening, neointimal lesion formation, elastin breakdown, increased anastomosis within the bronchial circulation, and perivascular inflammation. Using genetic lineage tracing, clonal analysis, multiplexed in situ hybridization, immunostaining, deep confocal imaging, and staged pharmacological inhibition, we define the cell behaviors underlying each stage of vascular remodeling and identify a pathway required for neointima formation. Results: Neointima arises from smooth muscle cells (SMCs) and not endothelium. Medial SMCs proliferate broadly to thicken the media, after which a small number of SMCs are selected to establish the neointima. These neointimal founder cells subsequently undergoing massive clonal expansion to form occlusive neointimal lesions. The normal pulmonary artery SMC population is heterogeneous, and we identify a Notch3-marked minority subset of SMCs as the major neointimal cell of origin. Notch signaling is specifically required for the selection of neointimal founder cells, and Notch inhibition significantly improves pulmonary artery pressure in animals with pulmonary hypertension. Conclusions: This work describes the first nongenetically driven murine model of pulmonary hypertension (PH) that generates robust and diffuse occlusive neointimal lesions across the pulmonary vascular bed and does so in a stereotyped timeframe. We uncover distinct cellular and molecular mechanisms underlying medial thickening and neointima formation and highlight novel transcriptional, behavioral, and pathogenic heterogeneity within pulmonary artery SMCs. In this model, inflammation is sufficient to generate characteristic vascular pathologies and physiological measures of human PAH. We hope that identifying the molecular cues regulating each stage of vascular remodeling will open new avenues for therapeutic advancements in the treatment of PAH.

Glioblastomas (GBM) are characterized by a strong immunosuppressive environment, contributing to their poor prognosis and limited therapeutic response to immunotherapies. B-cells represent a unique opportunity to promote immunotherapy due to their potential to kill tumors by both cellular and humoral immunity. To generate our B-cell-based vaccine (BVax) platform, we activated 41BBL+ B cells from tumor bearing mice or GBM patient blood with BAFF, CD40, and IFNg. We have previously demonstrated that BVax potentiates radiation therapy, temozolomide and checkpoint blockade in murine models of GBM via enhancement of CD8+ T-cell based immunity. The aim of this current study is to evaluate the humoral effector functions of BVax. We examined the antibody (Ab) repertoire in vivo from serum of tumor-bearing B-cell knockout mice treated with BVax or by ex vivo stimulation of patient-derived BVax. Upon systemic administration, BVax infiltrates the tumor where it differentiates into plasmablasts. Murine BVax- and BNaive-derived serum immunoglobulin generated in vivo showed that the majority of murine BVax-derived Ab were IgG isotype, while BNaive mainly produced IgM isotype. Transfer of IgG from BVax treated mice directly into tumors of recipient animals significantly prolonged their survival, demonstrating anti-tumor cytotoxicity directly through humoral immunity. Patient-derived BVax activated ex vivo showed a plasmablast phenotype and the Ab repertoire supports the previous findings seen in our murine model. Our work suggests BVax-derived IgGs role in antibody-dependent cellular cytotoxicity and improved survival in murine models. This function, in addition to its role in cellular immunity against GBM, renders BVax a potentially effective alternative immunotherapeutic option for GBM patients.

Malignant brain tumors are uniquely immunosuppressive, with a predominant infiltration of immunosuppressive tumor-associated myeloid cells (TAMCs) and a deficit in T-cells unrivaled to any other tumor. This unique tumor microenvironment (TME) promotes resistance to both conventional and immune therapies for this disease. The underlying mechanisms by which TAMCs promote glioblastoma (GBM) progression are not fully understood. We found that TAMCs specifically upregulate de-novo creatine metabolism within GBM using unbiased genetic and metabolic screening. This metabolic phenotype was confirmed in human GBM patients by comparing peripheral versus tumor-infiltrating myeloid cells. Examination of de-novo creatine generation using Carbon13 arginine flux revealed that TAMCs, but not tumor-infiltrating CD8+ T-cells, can produce creatine. Furthermore, we demonstrate that TAMCs actively secrete de-novo generated creatine into cell cultures. Examination of the single-cell microenvironment of GBM revealed that malignant cells preferentially express the creatine transporter, indicating that TAMC-derived creatine is taken up by GBM. Notably, SLC6A8 is directly upregulated in the context of hypoxia and suggests that creatine uptake is a mechanism to promote survival under hypoxic stress. Indeed, exogenous creatine supplementation promoted both the migration and survival of multiple glioblastoma cell lines in-vitro. Utilizing an established inhibitor of creatine metabolism, β-Guanidinopropionic acid (β -GPA), we found that β -GPA blocks both the migration and survival of glioma cells under hypoxic stress. Lastly, β -GPA also inhibited creatine secretion by TAMCs, showing that creatine blockade can also influence TAMC metabolic phenotype. In the future, we will examine the importance of creatine metabolism on both immune suppression and tumor progression in-vivo. This work provides novel insights into the role of creatine metabolism in GBM and identifies a unique therapeutic avenue for this devastating disease.

Simple Summary Glioblastoma is the most common primary brain tumor in adults, and its aggressive nature yields a poor prognosis despite current treatment strategies. The aim of our literature review is to discuss various immunotherapeutic strategies currently being investigated in the treatment of glioblastoma. Checkpoint inhibitors, various vaccination strategies, and CAR T-cell therapies serve as some of the most investigated immunotherapeutic strategies. However, all strategies face various limitations, such as the low relative mutational burden, the immunosuppressive tumor microenvironment, and genetic heterogeneity, which serve as the current challenges. Abstract Glioblastoma (GBM) is a lethal primary brain tumor. Despite extensive effort in basic, translational, and clinical research, the treatment outcomes for patients with GBM are virtually unchanged over the past 15 years. GBM is one of the most immunologically “cold” tumors, in which cytotoxic T-cell infiltration is minimal, and myeloid infiltration predominates. This is due to the profound immunosuppressive nature of GBM, a tumor microenvironment that is metabolically challenging for immune cells, and the low mutational burden of GBMs. Together, these GBM characteristics contribute to the poor results obtained from immunotherapy. However, as indicated by an ongoing and expanding number of clinical trials, and despite the mostly disappointing results to date, immunotherapy remains a conceptually attractive approach for treating GBM. Checkpoint inhibitors, various vaccination strategies, and CAR T-cell therapy serve as some of the most investigated immunotherapeutic strategies. This review article aims to provide a general overview of the current state of glioblastoma immunotherapy. Information was compiled through a literature search conducted on PubMed and clinical trials between 1961 to 2021.