bluuebunny/crossref_metadata_2025_split huggingface dataset:
IMMU-09. IMMUNOLOGICAL CHARACTERIZATION OF PEDIATRIC BRAIN TUMORS HAS CLINICAL IMPLICATIONS FOR PATIENT MANAGEMENT AND PROGNOSIS
Authors: Levine Adrian , Nobre Liana , Milos Scott , Das Anirban , Johnson Monique , Laxer Ben , Ryall Scott , Siddaway Robert , Tabori Uri , Hawkins Cynthia 
Published: November 2022

Abstract
The tumor immune microenvironment (TIME) is a growing area of interest, however, the extent of immune activation in childhood CNS tumors is unknown. Although immunotherapy has not gained success in most CNS cancers, our group described gliomas with MMRD which exhibit hypermutation and favorable responses to immune checkpoint inhibition (ICI). Therefore, detailed characterization of the CNS TIME is key for the development of novel immunotherapeutic strategies and application of existing ones in childhood brain tumors. We developed a clinical NanoString immune-oncology panel that includes markers reflecting cell types, therapeutic targets, and cellular pathways, as well as the 18-gene tumor inflammation signature (TIS), a biomarker for ICI response. We tested over 500 brain tumors, including 266 low-grade gliomas (LGG), 170 high-grade gliomas (HGG), 91 MMRD tumors, 16 ependymomas, 46 medulloblastomas, and 36 non-tumor brain samples. Overall, ependymomas and medulloblastomas had low levels of inflammation, although SHH medulloblastomas had higher inflammation than other subtypes. IDH-mutant LGG were immunologically cold, while many gliomas with pediatric-LGG mutations had high levels of inflammation, including upregulation of immune checkpoints – indicating that ICI may be an effective strategy. Interestingly, in pediatric-LGG inflammation impacted outcome in tumors with the same genetic alterations. BRAF V600E-mutant LGG exhibiting high TIS had inferior prognosis (p = 0.02), while no such relationship was observed in BRAF-fused tumors. Diffuse midline gliomas had higher inflammation than hemispheric HGG, indicating that these tumors are not immunologically cold, as has been previously reported. In MMRD tumors treated with ICI, high TIS correlated with improved survival and was independent from hypermutation and mutational burden. Furthermore, MMRD gliomas had high expression of several other immune checkpoints including LAG3, suggesting its value as an additional therapeutic target. In summary, characterization of the TIME across pediatric brain tumors provides potential prognostic clues and suggest treatment strategies for further investigation.
IMMU-13. PROMOTER DEMETHYLATION OF NY-ESO-1 PRIMES GLIOBLASTOMA FOR IMMUNOTHERAPEUTIC TARGETING
Authors: Lai Thomas , Treger Janet , Rao Jingyou , Sun Lu , Li Tie , Lai Albert , Prins Robert , Everson Richard G 
Published: November 2022

Abstract

INTRODUCTION
New York-esophageal squamous cell carcinoma (NY-ESO-1) specific adoptive T-cell immunotherapy is an appealing strategy for the treatment of gliomas because it elicits a strong antibody and T-cell response and is downregulated in most normal adult tissues which produces an effective and specific antitumor response. However, NY-ESO-1 is often poorly expressed in GBM, presumably through promoter methylation. Neither the prevalence of NY-ESO-1 downregulation in glioblastoma (GBM) patient tumors nor the presumed mechanism of downregulation by promoter methylation have been formally established.

METHODS
We characterized baseline CpG methylation of NY-ESO-1 in patient GBM samples using bisulfite sequencing. We induced NY-ESO-1 expression in vitro in human GBM cell lines (U251) and patient-derived gliomaspheres using the demethylating agent decitabine (DAC). We investigated the epigenetic response of DAC treated GBM cell lines with bisulfite sequencing and NY-ESO-1 expression with quantitative real time PCR. Lastly, we performed single cell RNA sequencing on DAC treated and untreated U251 and PGDSC to evaluate tumor subpopulations that upregulate NY-ESO-1 and co-expressed cancer testis antigens after DAC treatment.

RESULTS
NY-ESO-1 expression is associated with promoter methylation in the majority of GBM. Prolonged low dose of DAC explicitly demethylated the promoter region of NY-ESO-1 and resulted in robust mRNA expression. DAC treatment co-expresses NY-ESO-1 with other cancer testis antigens CTAG2 and MAGEA4 as demonstrated by single cell RNA sequencing.

CONCLUSION
These results reveal a feasible strategy of cancer testis antigen promoter demethylation which creates an inducible tumor antigen for immunotherapeutic targeting. We speculate that DAC induced upregulation of NY-ESO-1 and co-expressed cancer testis antigens renders tumor recognition by NY-ESO-1 specific T cells.
IMMU-14. MENINGEAL PLASMA CELLS MOUNT AN IMMUNOLOGICAL DEFENSE AGAINST GLIOBLASTOMA
Authors: Ampie Leo , McGavern Dorian 
Published: November 2022

Abstract
INTRODUCTION: Glioblastoma (GBM) represents the deadliest primary intracranial neoplasm. While many studies have focused on T cell responses to GBM, few have examined the role of humoral immunity. Here, we sought mechanistic insights into the anatomy and function of the humoral response against GBM. METHODS: C57BL/6J mice were injected stereotactically with syngeneic SB28 tumor cells. Humoral immunity was evaluated in the spleen and blood by high parameter flow cytometry and the meninges, brain, and spleen by confocal microscopy. The function importance of humoral immunity was interrogated using CD4-/- and IgA-/- mice and via transcranial application of the plasma cell survival factor, APRIL. RESULTS: High parameter flow cytometry revealed a significant increase of plasma B cells (CD19+, CD138+) in the spleen and blood of GBM mice within two weeks. Using blimp-1 reporter mice, we observed that IgA+ plasma cells localized to extrafollicular splenic regions and along meningeal dural venous sinuses near newly generated blood and lymphatic vessels that connected directly to the tumor. Decreased survival was observed in IgA-/- but not CD4-/- mice injected with SB28 cells, suggesting that a T helper independent IgA response plays a role in controlling GBM. This response was enhanced therapeutically by transcranial application of APRIL. CONCLUSION: Our results demonstrate that GBM elicits a systemic and meningeal humoral response, which plays in limiting GBM growth and can be transcranially enhanced via APRIL. The participation of IgA+ plasma cells in GBM immunity offers new possibilities for therapeutic intervention in this deadly disease.
IMMU-15. HUMAN ALLOGENIC γδ T CELLS KILL PATIENT-DERIVED GLIOBLASTOMA CELLS IN A PVR- AND DNAM-1-DEPENDENT MANNER
Authors: Ahn Stephen , Choi Haeyoun 
Published: November 2022

Abstract

Background
Adoptive transfer of γδ T cells is a novel immunotherapeutic approach to glioblastoma. Few recent studies have shown the efficacy of γδ T cells against glioblastoma, but no previous studies have identified the ligand–receptor interactions between γδ T cells and glioblastoma cells. Here, we identify those ligand–receptor interactions and provide a basis for using γδ T cells to treat glioblastoma.

METHODS
Vγ9Vδ2 T cells were generated from peripheral blood mononuclear cells of healthy donors using artificial antigen presenting cells. MICA, ULBP, PVR and Nectin expression in 10 patient-derived glioblastoma (PDG) cell lines were analyzed. The in vitro cytokine secretion from the γδ T cells and their cytotoxicity toward the PDG cell lines were also analyzed. The in vivo anti-tumor effects were evaluated using a U87 orthotopic xenograft glioblastoma model.

RESULTS
Expression of ligands and cytotoxicity of the γδ T cells varied among the PDG cell lines. Interestingly, cytotoxicity correlated significantly with the PVR expression of the PDG cells. IFN-γ and granzyme B secretion levels were significantly higher when γδ T cells were co-cultured with high PVR–expressing PDG cells than when they were co-cultured with low PVR–expressing PDG cells. Blocking DNAM-1 resulted in a decrease in γδ T cell–mediated cytotoxicity and cytokine secretion. Intratumoral injection of γδ T cells showed anti-tumor effects in an orthotopic mouse model.

Conclusions
Allogenic γδ T cells showed potent anti-tumor effects on glioblastoma in a PVR- and DNAM-1-dependent manner. Our findings will facilitate the development of clinical strategies using γδ T cells for glioblastoma treatment.
IMMU-16. SOMATIC MUTATIONS IN HUMAN LEUKOCYTE ANTIGEN GENES AND ANTIGEN PRESENTATION PATHWAY GENES IN MALIGNANT GLIOMA
Authors: Schulte Sara , Trojok Peter , Schäfer Moritz , Peter Wolfgang , Westphal Manfred , Lamszus Katrin , Dilthey Alexander , Mohme Malte 
Published: November 2022

Abstract

Immunotherapeutic approaches in cancer aim to booster T cell mediated immune responses. Tumor-specific T cells recognize target cells via human leukocyte antigen (HLA) molecules before cell specific lysis is initiated. Malignant gliomas are known for their immunoevasive and immunosuppressive properties which impede effective tumor-specific immune responses, including clinical responses to immune checkpoint inhibition. Our aim was to investigate whether somatic mutation in HLA genes and antigen presentation pathway genes are present in malignant gliomas.

METHODS
We performed next generation sequencing in 138 patients, including matched tumor and blood samples from 100 IDHwt glioblastomas, 25 IDHmut astrocytomas and 13 IDHmut oligodendrogliomas. Sequencing was performed on tumor and peripheral blood for HLA class I (HLA-A, -B, -C), HLA class II (HLA-DR, -DP, -DQ), non-classical HLA molecules (HLA-E, -F, -G, -H), MICA/B, TAP1/2 and beta2 microglobulin (B2M). Identified calls were validated using targeted nanopore sequencing.

RESULTS
In total, we identified 28 mutations based on Illumina sequencing. Mutations were located in HLA-B (2 mutations), HLA-C (1), B2M (2), HLA-DQA1 (4), HLA-DQB1 (4), HLA-DPB1 (1), HLA-E (1), HLA-F (2), HLA-G (3), HLA-H (1), MICA (3), MICB (2), TAP1 (1) and TAP2 (1). No mutations were found in HLA-A and HLA-DRB1. A frequency cutoff of 5% was applied. Additional mutations (n = 46) below this cutoff were identified. The mutations were found in 30 patients (21.7%). Some patients harbored up to 7-8 mutations in multiple HLA and antigen presenting pathway genes. Most mutations were found in the MICA (MHC class I polypeptide–related sequence A) gene locus, which is engaged by the NKG2D ligand which is broadly expressed on NK cells, gd and ab T cells.

CONCLUSION
Our study demonstrates that somatic mutations in HLA and antigen presentation pathway genes are frequently present in malignant gliomas.
IMMU-18. MIGRATION OF DENDRITIC CELLS THROUGH THE BRAIN-MENINGEAL LYMPHATIC-DRAINING LYMPH NODE NETWORK IN ORTHOTOPIC GLIOMA MODELS
Authors: Batich Kristen , Chen Ching Wen , Wellford Sebastian , Dao Kianna , Moseman Annie Park , Hotchkiss Kelly , Cook Sarah , Snyder David , Sampson John , Moseman Ashley 
Published: November 2022

Abstract

INTRODUCTION
Limited migration of dendritic cell (DC) vaccines to draining lymph nodes (DLN) remains a major limitation to DC efficacy for malignant gliomas. Our prior work demonstrated that increased peripheral DC migration to DLN results in enhanced antitumor efficacy. Given this, we studied DC trafficking in mouse gliomas, the meningeal lymphatic vessel (MLV) system, and the draining cervical lymph nodes (CLN).

METHODS
To elevate host DC populations, C57BL/6 and VMDk mice were implanted with B16-FMS-like tyrosine kinase 3 ligand (FLT3L) melanoma or with B16-FLT3L supernatant. Mice were implanted intracranially with CT2A, GL261-OVA, and SMA560 syngeneic glioma lines in the right parietal lobe. Antitumor efficacy was measured by tumor weights and median overall survival (mOS).

RESULTS
DC injected into glioma hemispheres display delayed kinetics of peak migration to CLN compared to control (7 days (d) vs 3d post-injection (pi), respectively). Migration of intraventricular (ivn)-injected DC to CLN is superior at days 1, 3 and 7 pi when glioma is present (p = 0.026). B16-FLT3L supernatant results in a disproportionate systemic expansion in conventional type 1 over type 2 DC (cDC1 &gt; cDC2) compared to media control (cDC1, cDC2 fold change over media for spleen = 27,11; DLN = 6,2; blood = 6,3). Pulsed dosing of FLT3L supernatant results in significantly greater numbers of splenic cDC1 compared to daily FLT3L dosing (p = 0.002). Mice with CT2A and continuous FLT3L secretion in vivo show longer survival (mOS 30d) compared to mice with continuous GM-CSF secretion (mOS 22d) (p = 0.009). Mice with SMA560 and FLT3L secretion have significantly reduced glioma growth compared to tumor alone, GM-CSF-secreting tumors, and mixed FLT3L/GM-CSF-secreting tumors (p = 0.029).

CONCLUSION
FLT3L signaling in vivo results in cDC1 &gt; cDC2 generation and migration through the CNS with more favorable glioma efficacy. Modeling of DC migration through the glioma-MLV-CLN system with FLT3L stimulation permits testing of strategies to improve DC therapy.
IMMU-21. GLIOMA-DERIVED FACTORS RECRUIT AND INDUCE AN IMMUNE SUPPRESSIVE PHENOTYPE IN BONE MARROW-DERIVED CCR2+ MYELOID CELLS
Authors: Takacs Gregory , Kreiger Christian , Luo Defang , Tian Guimei , Deleyrolle Loic , Harrison Jeffrey 
Published: November 2022

Abstract

INTRODUCTION
Infiltrating immune-suppressive myeloid cells represent a tumor supportive population that contributes to immune checkpoint inhibitor resistance and poor survival in Glioblastoma (GBM) patients. We have previously characterized monocytic-myeloid derived suppressor cells (M-MDSCs) based on their dual expression of chemokine receptors CCR2 and CX3CR1(CCR2+/CX3CR1+). Genetic and pharmacologic targeting of CCR2, in combination with PD-1 blockade, reduced the percentage of M-MDSCs in the glioma-microenvironment and slowed the progression of KR158 and 005GSC murine gliomas. Additional studies are needed to investigate the chemokines responsible for the tumor recruitment of CCR2+/CX3CR1+ cells and the impact of glioma derived factors on their immune suppressive phenotype.

OBJECTIVE
Evaluate the effect of glioma derived factors on the migration and suppression of bone marrow CCR2+/CX3CR1+ myeloid cells.

METHODS
A transwell migration assay was utilized to determine the migratory ability of CCR2+/CX3CR1+ cells to KR158B conditioned in the presence of CCL2 and CCL7 neutralizing antibodies. Ly6G-/GR1+ cells were isolated from bone marrow cultured with KR158B conditioned media and co-cultured with freshly isolated T-cells to examine their immune-suppressive phenotype.

RESULTS
KR158B gliomas differentially upregulate cytokines including CCL2, IL6, G-CSF, GM-CSF as compared to healthy naive brains. KR158B conditioned media increased the percentage of bone marrow-derived CCR2+/CX3CR1+ cells that are CD11b+, Ly6Chi, and Ly6G-. Bone marrow-derived CCR2+/CX3CR1+ cells expanded in KR158B condition media suppress both CD4+ and CD8+ T cell proliferation. Bone marrow-derived CCR2+/CX3CR1+ cells migrate to recombinant CCL2 and CCL7 as well as KR158B glioma conditioned media. Migration to conditioned media is completely inhibited by the combination of CCL2 and CCL7 neutralizing antibodies. High CCL2 and CCL7 are associated with poor prognosis in human GBM.

CONCLUSION
Glioma-derived CCL2 and CCL7 mediate migration of CCR2+ myeloid cells into the tumor microenvironment in a redundant manner. Additional glioma-derived factors induce CCR2+/CX3CR1+ myeloid cells to a CD4/8+ T cell suppressive state.
IMMU-27. SIMULTANEOUS TARGETING OF THE PD-L1 AND THE TGF-Β PATHWAYS WITH BINTRAFUSP ALFA IN EXPERIMENTAL GLIOMAS
Authors: Silginer Manuela , Weller Michael , Kienzler Jenny , Nunez Nicolas , Lan Yan , Weiss Tobias , Becher Burkhard , Roth Patrick 
Published: November 2022

Abstract
Currently, immunotherapy is one of the most promising research areas in oncology, but immunotherapeutic strategies do not have proven clinical activity against glioblastoma so far. The immunosuppressive microenvironment of gliomas limits an appropriate anti-tumor immune response and represents a major challenge in the context of immunotherapy. Therefore, novel therapeutic strategies that reactivate the immune system and allow for potent anti-glioma immune responses are needed. Bintrafusp alfa is a bifunctional fusion protein that is composed of the extracellular domain of human transforming growth factor-beta receptor type II (TGF-βRII) and a human IgG1 monoclonal antibody blocking programmed death-ligand 1 (PD-L1). It is designed to target the TGF-β and the PDL-1 pathways simultaneously by specifically localizing to the tumor via the anti-PD-L1 moiety and sequestering TGF-β within the tumor microenvironment. We analyzed interrelations between the immunosuppressive factors TGF-β and PD-L1 in glioblastoma samples from patients and observed a significant correlation that points towards a co-regulation of both targets as part of a gene expression network. Next, we assessed the activity of bintrafusp alfa in human and murine glioma cell lines in vitro and observed interference with TGF-β-induced Smad2 phosphorylation as a bona fide response marker of TGF-β pathway activation and an increase in immune cell-mediated glioma cell killing. In vivo, bintrafusp alfa prolonged the survival of glioma-bearing immunocompetent mice, whereas the survival benefit was absent in immunodeficient Rag1-/- mice lacking T and B cell populations. Characterization of the tumor microenvironment by immunohistochemistry and high-dimensional flow cytometry confirmed the immune-activating effects of bintrafusp alfa. In summary, these encouraging results demonstrate that novel treatment options such as bintrafusp alfa may have the potential to overcome the lack of immunogenicity and reinvigorate anti-tumor immune responses to efficiently combat glioblastoma.
IMMU-29. PERIPHERAL VACCINATION WITH GIFT-7 SECRETING TUMOR CELLS AUGMENTS AGE RELATED THYMIC DECLINE AND IMPROVES SURVIVAL ACROSS MULTIPLE GLIOBLASTOMA MOUSE MODELS
Authors: Shireman Jack , Zhao Lei , Wang Xiaohu , Galipeau Jacques , Dey Mahua 
Published: November 2022

Abstract
Glioblastoma (GBM), the most common primary brain tumor in adults, has a dismal prognosis and nearly 100% recurrence rate. With a median age of diagnosis of 64, age associated immune dysfunction needs to be accounted for during treatment. Cannonically, immunotherapies target checkpoint signaling pathways in order to create T-cell populations that are resistant to exhaustion. Our lab has demonstrated that exhaustion is not the only factor at play regarding T-cell dysfunction, as patients with GBM have a robust CD8+CD28- effector population present both in the blood and tumor, possibly indicating T-cell senescence. Here we present research utilizing GIFT-7, a fusiokine combining GMCSF and IL-7, which has been demonstrated to combat age related thymic decline. To accurately understand this phenomenon in the context of aging all experiments were done in mice aged 52 weeks before intial tumor injection. We transfected the mouse glioma line GL261 cells to stably express and secrete GIFT-7 and injected 500k cells (GIFT7/VC) subcutaneously in the flank of B6 mice. This flank tumor allowed both for continued systemic administration of GIFT7, as well as immune exposure to the tumor similar to a vaccine model. After 4 weeks an intracranial challenge of normal GL261 cells was conducted and survival was monitored. Mice with GIFT-7 flanks had a larger population of CD3 and CD4 T-cells in their systemic circulation (p &lt; .001 and p &lt; .05 respectively). We also observed that the GIFT-7 flank mice demonstrated an increased tendency to have T-cells of all populations (CD3, CD4+, and CD8+) present within the brain. Finally, GIFT-7 flank mice survival was vastly improved with 60% of mice showing long term survival ( &gt; 120 days) compared to 0 VC mice.
IMMU-30. THE ROLE OF CD4 T CELLS IN ANTITUMOR IMMUNITY AGAINST BRAIN TUMORS
Authors: Coxon Andrew , Desai Rupen , Livingstone Alexandra , Kobayashi Dale , Dunn Gavin , Johanns Tanner 
Published: November 2022

Abstract
Glioblastoma is a primary brain tumor with a dismal prognosis. CD4 T cell responses to neoantigen vaccines or checkpoint blockade immunotherapy impact the tumor microenvironment. However, the function of CD4 T cells in glioblastoma remains understudied. Here we further characterize the role of CD4 T cells in antitumor immunity against the GL261 orthotopic model of glioblastoma. We studied the memory response generated during high-dose PD-L1 blockade mediated clearance of intracranial tumors. Mice that previously rejected GL261 retained protection against intracranial rechallenge after depletion of CD4 or CD8 T cells, but not both. Thus, these memory CD4 T cells are sufficient but not necessary for protection. Interestingly, in mice with a memory response generated during clearance of extracranial GL261, depletion of CD4 T cells abrogates protection against intracranial rechallenge, but depletion of CD8 T cells alone does not affect protection. Therefore, memory CD4 T cells generated with extracranial antigen challenge are necessary and sufficient for protection against intracranial rechallenge. In an analogous setting, we vaccinated mice against MHC class I neoantigens with concurrent checkpoint blockade immunotherapy to generate memory responses. Similar to the challenge-rechallenge model, mice challenged with GL261 and depleted of CD4 T cells lost protection from neoantigen vaccination and PD-L1 blockade. We then modified GL261 to express the C-terminal end of ovalbumin (GL261-OVAII), which contains the MHC class II immunodominant antigen (OVA323-339) but not the class I antigen (OVA257-264). Mice challenged intracranially with GL261-OVAII have a minimal survival benefit from PD-L1 blockade therapy. However, vaccination against OVA323-339 greatly enhances the protection from PD-L1 blockade. We determined by ELISPOT that CD4 T cells do not directly recognize GL261. Our data show the importance of the CD4 T cell subset in glioblastoma and the importance of targeting class II neoantigens to generate strong antitumor immunity.
IMMU-33. CCL4 DRIVES GLIOBLASTOMA (GBM) TUMOR MICROENVIRONMENT (TME) REPROGRAMMING AFTER TREATMENT WITH A NOVEL MRNA VACCINE
Authors: Dastmalchi Farhad , Thomas Nagheme , Frain Matt , Ebrahim Ghaidaa , Moore Ginger , Tomdio Macaulay , Mendez-Gomez Hector , Sayour Elias , Subramaniam Vignesh , Angelini Thomas , Mitchell Duane , Rahman Maryam 
Published: November 2022

Abstract

INTRODUCTION
Our group has developed a patented vaccine platform that combines an mRNA-nanoparticle with CXCL9 loaded polyethylene glycol (PEG) hydrogel. The HCM vaccine is given SQ and recruits a diverse immune cell population to deliver a large payload of mRNA. The objective of these studies was to evaluate the effects of HCM vaccination on the TME.

METHODS
C57BL/6 mice underwent intracranial implantation of KR158 or GL261 glioma cells. The HCM vaccine was formulated with total tumor mRNA, DOTAP, CXCL9 and PEG hydrogel created in collaboration with the College of Engineering. Survival studies were analyzed with the Mantel-Cox test. Flow cytometry and ELISA data was analyzed using ANOVA. Graphpad was used for statistical analysis.

RESULTS
Intracranial tumor bearing mice were treated with SQ or intracranial injection of the HCM vaccine (total tumor mRNA, DOTAP, CXCL9, PEG hydrogel). SQ HCM delivery resulted in significant survival benefit whereas intracranial delivery did not (p = 0.0012). In a separate experiment, tumors and spleens were collected for flow cytometry. A significant increase in antigen specific CD8 T cells was found. Separately, after SQ vaccination, tumors were collected for PCR analysis. Tumors treated with HCM vaccination demonstrated significant upregulation of CCL4 and CXCR6. Intracranial tumor bearing mice were treated with HCM vaccination with or without blockade of CCL4. CCL4 blockade resulted in no survival benefit of the vaccine.

CONCLUSION
The HCM vaccine results in migration of antigen specific CD8 T cells within the TME and significant increases in CCL4. HCM vaccine efficacy is dependent on CCL4.