bluuebunny/crossref_metadata_2025 huggingface dataset:
IMMU-01. DEFINING THE IMMUNE PROFILE OF RADIATION NECROSIS THROUGH SINGLE-CELL ANALYSIS OF INTRACRANIAL LESIONS
Authors: Robert Stephanie , Lu Benjamin , Kiziltug Emre , Estape Anna , Nguyen Don , Chiang Veronica
Published: November 2022

INTRODUCTION

The incidence of radiation necrosis (RN), a common and morbid side effect of radiosurgical management of brain metastases, is growing as patient survival and use of immunotherapy increases. RN is thought to be immunologically driven; however, its pathophysiology remains poorly understood. Further, difficulty distinguishing it radiographically from recurrent metastatic disease often necessitates invasive brain biopsies for diagnosis. We sought to define the neuroimmune profile of RN to better understand the underlying pathophysiological mechanisms and improve patient diagnosis.

METHODS

Over 12 months, we collected intraoperative samples from patients undergoing craniotomy for RN and/or metastatic tumor. We created a bank of these 16 patient samples and processed them for scRNAseq analysis. Using fluorescence-activated cell sorting (FACS) and 10x Genomics single cell RNA sequencing, we analyzed the cellular immune profile of lesional tissue and time-matched blood from each patient.

RESULTS

Compared to metastases, RN demonstrates decreased expression of FOXP3, NKG7, and GZMB, markers of regulatory and cytotoxic T, and natural killer (NK) cells, and increased expression of interleukin-7 receptor (IL7R), a marker of naïve and memory T cells – profiles reflective of bystander transcriptional signatures. In contrast, metastatic tissue demonstrates an antigen-reactive profile, including commonly observed exhaustion signatures. Importantly, blood samples from patients also reflect these differences. Interestingly, increased IL7R is implicated in autoimmune disorders, suggesting an iatrogenically-induced autoreactive process underlying RN.

DISCUSSION

These findings demonstrate, firstly, the feasibility of extracting live cells from RN tissue, and, secondly, suggest RN and metastases have unique immune profiles that can be detected in patient blood. Therefore, in addition to beginning to define the immune landscape of this poorly understood pathology, this work has the potential to open new avenues for the development of improved diagnostic and therapeutic approaches to allow for earlier detection and more effective treatment of RN in metastatic brain tumors patients.
IMMU-07. HUMANIZED BI-SPECIFIC ANTIBODY SHOWS POTENT ACTIVITY IN PATIENT-DERIVED XENOGRAFT MODELS OF GLIOBLASTOMA
Authors: Duffy Joseph , Zannikou Markella , Levine Rebecca , Stupp Roger , James David , Balyasnikova Irina
Published: November 2022

BACKGROUND

Advancements in bioengineering have allowed bispecific Abs to develop with therapeutic applications in immuno-oncology. Here we describe a fully humanized bispecific t cell engager (BiTE) that binds the GBM-associated antigen interleukin 13 receptor alpha 2 (IL13Rα2) and the T Cell receptor CD3 epsilon (CD3ε) to elicit an anti-tumor immune response in patient-derived models of GBM.

METHODS

In vitro, BiTE-mediated activation of T Cells was assessed via flow cytometry as measurements of T cell activation markers CD25 and CD69 and cytokines IFNγ and TNFα. BiTE-directed T cell-mediated cytotoxicity was assessed in GBM 6 and GBM12patient-derived xenograft lines. In vivo, GBM6 patient-derived xenograft cells were intracranially implanted in NSG-MHC I/II DKO mice, reconstituted with HLA-matched donor peripheral blood lymphocytes (PBMC), and subsequently treated with 2.5 mg/kg BiTE via i.p. injection x 4 days/week of therapeutic BiTE for three weeks. Animals were followed for survival, and the brains of long-term survivors were analyzed for residual disease and T cell infiltration.

RESULTS

In vitro, BiTE potently activated T cells as determined by a high expression of CD25 and CD69 and the production of TNFα and IFNγ. BiTE also directed T cell-mediated cytotoxicity against GBM cells in a dose-dependent manner, with significant activity still seen in the low nanogram range. In vivo, BiTE significantly extended the survival of tumor-bearing animals in GBM6patient-derived xenograft models. Histological analysis of long-term surviving mice shows complete absence of tumor and robust presence of CD8+ T cells localized to the original site of tumor implantation.

CONCLUSIONS

Our data demonstrate the efficacy of humanized BiTE in patient-derived GBM models, and our results support the investigation of this novel therapeutic in a clinical trial setting.FUNDING: R01 NS122395-01 and R01 NS095642-01
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

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

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

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

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

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

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 > 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 > 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

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

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

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 < .001 and p < .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 ( > 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

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

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.
IMMU-34. NL-201, A DE NOVO ENGINEERED IL2 MIMETIC, SYNERGIZES WITH RADIOTHERAPY TO GENERATE POTENT ANTITUMOR ACTIVITY IN PRECLINICAL GLIOBLASTOMA MODELS
Authors: Huntoon Kristin , Lee DaeYong , Lu Yifei , Gallup Thomas , Jiang Wen , Kim Betty
Published: November 2022

Combining radiotherapy (RT), a standard of care treatment for many cancer types, with immunotherapeutic agents has been gaining interest due to improved efficacy. One such approach is RT in combination with high-dose recombinant interleukin 2 (IL-2), which elicits anti-tumor immune responses by stimulating T-cell and NK cell populations. Although effective for some patients, the overall clinical benefit of recombinant IL-2 is limited by toxicity at high doses and the expansion of regulatory T cells (Tregs) at low doses, effects thought to be mediated by preferential binding to IL2Rα (i.e. CD25). In this study we examined RT in combination with NL-201, a highly potent and stable CD25-independent IL-2/IL-15 agonist with enhanced affinity for the IL-2Rβγ heterodimeric receptor. Single-agent NL-201 was well tolerated in mice, yielded tumoricidal activity, expanded peripheral T cells, and enhanced the infiltration of effector T cells and dendritic cells (DCs) into murine glioblastoma (GL261 and SB28). In combination with RT, NL-201 enhanced activation of the cytosolic DNA sensor cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, resulting in increased type I interferon (IFN) production in DCs and, consequently, greater tumor infiltration by T cells and more efficient priming of antigen-specific T cells by professional antigen presenting cells (APCs). The immune stimulatory mechanisms triggered by the NL-201 and RT combination resulted in superior tumor growth inhibition. We demonstrated that the CD25-independent IL-2 and IL-15 receptor agonist, NL-201, in combination with RT is well-tolerated and elicits robust anti-tumor activity through both innate and adaptive responses, including in checkpoint resistant tumors. Furthermore, we identify a unique mechanism in which NL-201 synergizes with RT. Taken together, the results provided herein support further preclinical and clinical investigation of this novel combination regimen.
IMMU-37. ANTI-CD47 TREATMENT POTENTIATES PHAGOCYTOSIS OF MEDULLOBLASTOMA BY M1 POLARIZED MACROPHAGES FOLLOWING IRRADIATION
Authors: Gamboa Nicholas , Baker Cordell , Youssef Osama , Cheshier Samuel
Published: November 2022

Medulloblastoma (MB) is the most common malignant brain tumor of childhood and accounts for 15–20% of all pediatric brain tumors. Despite significant advancements over the last decade in our understanding of the molecular underpinnings of MB, the overall risk of recurrence remains high, prognosis remains poor, and available treatment modalities remain limited for patients with this disease. Accordingly, the area of targeting tumor immune resistance has garnered increasing interest as a potentially safe and effective adjunctive therapy. CD47 is a cell surface protein that acts as a primary “don’t eat me” signal to the innate immune system and has been shown to be crucial in allowing tumor cells to evade phagocytosis by tumor-associated macrophages. Several preclinical studies aimed at blocking this “don’t eat me signal” through the application of a mouse anti-human monoclonal antibody (Ab) to CD47 (anti-CD47) have shown promise in pediatric brain tumor models. Further, chemotherapy and irradiation have been shown to increase the expression of pro-phagocytic signals on the surfaces of tumor cells. The authors examined the use of anti-CD47 Ab on the rates of phagocytosis of MB cells undergoing different single-fraction doses of external beam irradiation in vitro. The use of anti-CD47 Ab in the D425 MB cell line phagocytosis assay significantly increased rates of phagocytosis for 0 Gy (4.1% without vs. 20.3% with anti-CD47 Ab; p < 0.0001), 2 Gy (7.4% without vs. 23.4% with anti-CD47 Ab; p < 0.0001), 5 Gy (11.9% without vs. 27.3% with anti-CD47 Ab; p < 0.0001), 10 Gy (21.7% without vs. 34% with anti-CD47 Ab; p < 0.0001), and 20 Gy treatment groups (27.2% without vs. 43.9% with anti-CD47 Ab; p < 0.0001). A similar pattern of phagocytosis potentiation through use of anti-CD47 Ab with increasing doses of radiation was seen in D458 and D341 MB cell lines.
IMMU-40. KINETICS OF DEXAMETHASONE-INDUCED ALTERATIONS OF DIVERSE IMMUNE SUBSETS IN VIVO
Authors: Yaghi Nasser , Watanabe Masashi , Watowich Matthew , Li Aiguo , Briceno Nicole , Yaghi Omar , Livak Ferenc , Siddiqui Shafiuddin , Wolcott Karen , Zhang Wei , Zhang Meili , Song Hua , Davis Dionne , Gilbert Mark
Published: November 2022

INTRODUCTION

Immune therapeutic strategies are ideal for exploiting glial tumor heterogeneity. A limitation to their implementation for intra-axial brain tumors has been corticosteroids. Steroid use is associated with worse progression-free and overall survival when combined with some immunotherapies. Dexamethasone remains first line treatment for vasogenic cerebral edema and a primary therapy for immune-related adverse events secondary to immunotherapies. While used for decades, steroid immunosuppressive effects remain insufficiently characterized. We examined the temporal effects of dexamethasone on an extensive repertoire of immune cell subtypes.

METHODS

Non-tumor bearing C57BL/6 mice were treated with a onetime dose of 1 mg/kg dexamethasone via gavage, and peripheral blood was sampled at timepoints spanning 1 hour to 7 days. Flow cytometry was performed in triplicate to determine absolute cell counts of circulating B cells, CD8+ T cells (naive/memory), CD4+ T cells (naive/memory/regulatory), NK cells, neutrophils, monocytes, and eosinophils at each timepoint. We examined T cell function at multiple timepoints using an anti-CD3/CD28 bead stimulation assay.

RESULTS

At 1-hour post-treatment, cell counts across lymphoid subtypes increased. Neutrophils dramatically increased, while the remaining myeloid subtypes began to decrease. NK cells, similarly, were decreased in number. At 4-hours post-treatment all immune subtypes had their lowest circulating counts. There were corresponding increases in CD8+ and CD4+ T cell expansion and proliferation indices at 4-hours. By 24-hours post-treatment, all immune subtypes had recovered to or surpassed pre-treatment counts, except monocytes. By 7 days, the Tregs increased to 186% of pre-treatment counts, the highest of all subtypes, while eosinophils, monocytes, and NK cells never fully recovered to their pre-treatment baselines.

CONCLUSIONS

Understanding dexamethasone influence on circulating immune cells will inform optimal treatment timing for immunotherapies in brain tumor patients. We are further characterizing transcriptomic changes within immune cell subtypes following dexamethasone administration via population-level RNA sequencing over time.
IMMU-42. ADOPTIVE HEMATOPOIETIC STEM CELL AND APD1 THERAPY CONVERGE IN THE THYMUS TO PROMOTE ANTI-GLIOBLASTOMA IMMUNITY
Authors: Francis Connor , Font Laura Falceto , Figg John , Dean Bayli DiVita , Sebastian Mathew , Jin Dan , Long-James Kaytora , Von Roemeling Christina , Reid Alexandra , Moore Ginger , Mitchell Duane , Flores Catherine
Published: November 2022

Our lab previously published that adoptive hematopoietic stem cell (HSC) therapy administered in conjunction with aPD1 increases median survival in multiple models of CNS malignancies. Tumor-infiltrating lymphocytes (TIL) abundance is independently associated with better outcomes in many cancers. Impressively, approximately 25% of TILs are HSC-derived in early disease. Importantly, we show HSC therapy generates de novo TILs in a thymus-dependent fashion and offer evidence of altered central tolerance following aPD1 therapy, which may permit immune activation against a larger pool of tumor-associated antigens. Therapeutic HSCs were prepared from constitutive GFP-expressing (CD45.2+) and PD1KO (CD45.2+) mice and transferred into CD45.1+ hosts. Sublethal 5Gy total body irradiation was administered day 5 post-intracranial tumor implantation. The next day, 1E6 HSCs from a 1:1 – GFP:PD1KO cocktail were injected intravenously. Descriptive statistics are reported as mean ± SD. Transferred HSCs engraft, expand, differentiate, and migrate to form approximately 25% of the TIL compartment in only 15 days. We found that, 56.2% ± 20.3 of CD3+ TILs display a thymocyte-like CD4+CD8+ (DP) phenotype in early disease, and 43.8% ± 17.4 of these DP-TILs are HSC-derived. Interestingly, DP-TILs were virtually absent in advanced disease 29 days after treatment. At this stage, HSC-derived TILs most commonly fell within the single-positive (SP) TIL subsets, comprising 9.8% ± 5.6 of SP-CD8+ TILs. Notably, HSCPD1KO-derived cells were 4x more abundant amongst SP-CD8 TILs when compared to HSCGFP-derived cells (6.5% vs 1.6%, p < 0.05). SP-CD8 thymocytes were similarly enriched for HSCPD1KO progeny (41.1% vs 24.7%, p < 0.05) which may suggest PD-1 blockade directly promotes thymopoiesis. The privileged maturation of PD1-deficient HSCs is intriguingly reversed when GFP:PD1KO HSCs are co-transferred into non-tumor-bearing mice. Thus, we show the thymus is an active site where PD-1-blocking agents function, and that HSC + aPD1 therapy is uniquely positioned to leverage thymus-dependent anti-tumor immunity.
INNV-08. CASE REPORT OF AN OLDER GLIOBLASTOMA PATIENT SURVIVING MORE THAN 12 YEARS AFTER TREATMENT WITH CILENGITIDE IN ADDITION TO STANDARD THERAPY
Authors: Alalami Huda , Hu Jethro , Fan Xuemo , Bannykh Serguei
Published: November 2022

BACKGROUND

Glioblastoma is the most common malignant brain tumor. Less than 1% of patients survive longer than 10 years. Integrins are implicated in tumorigenesis due to their role in cell adhesion and intercellular communication. Cilengitide, a selective αvβ3, and αvβ5 integrin inhibitor, was studied for patients with glioblastoma.

RESULTS

A 77-year-old woman was found to have a left temporoparietal MGMT methylated glioblastoma after presenting with visual field deficits in 2009. She underwent near gross total resection of the tumor. The tumor had retained expression of PTEN and MAPK. No mutations in IDH or ATRX were detected. Genetic testing revealed mutations in TERT promoter, TP53, SYNE1, NF1, PTEN, FBXW7, POLD1, ERG, and PRKDC. She was enrolled in the CENTRIC trial, a phase III clinical trial studying the addition of cilengitide to standard therapy for patients with newly diagnosed glioblastoma with methylated MGMT promoter. In 2013, the initial results of the study failed to meet the endpoint of prolonged overall survival and progression-free survival, therefore the study was halted. The patient received cilengitide through compassionate use until 2016. Surveillance brain MRI obtained every three shows no radiographic evidence of recurrence.

CONCLUSION

Although it is known that methylated-MGMT promoter is one of the few positive prognostic factors in GBM, overall survival remains poor in older patients. This case highlights a rare clinical response to treatment in an older female who has survived more than 12 years without disease recurrence after receiving cilengitide in addition to standard therapy. Her outcome may have been influenced by the expression of PTEN and the methylation of MGMT, which are associated with improved outcomes. Although the development of cilengitide was halted for lack of efficacy, the existence of individual responders such as this patient suggests that further study of integrin inhibition for glioblastoma patients may be worth pursuing.
INNV-11. EIGHTEEN YEARS OF EXPERIENCE WITH TREATMENT OF GLIOBLASTOMA USING TUMOR TREATING FIELDS (TTFIELDS) IN NEWLY DIAGNOSED GLIOBLASTOMA (NDGBM) IN A SINGLE CENTER
Authors: Vymazal Josef , Novák Tomáš , Slanina Petr , Kazda Tomáš , Sroubek Jan , Klener Jan , Hrbáč Tomáš , Syrucek Martin , Rulseh Aaron
Published: November 2022

Despite considerable progress in the management of many malignant tumors, the prognosis of the most malignant primary brain tumor, GBM, remains unfavorable. TTFields utilize low intensity electric fields with intermediate frequency (100–500 kHz) that act as an antimitotic, selectively inhibiting growth of rapidly dividing tumor cells by disrupting multiple phases of the cell cycle (metaphase, anaphase, and telophase) resulting in cellular apoptosis. TTFields are delivered via arrays of transducers placed on the patients’ scalp. Between 2004 and August 2021, 54 patients (KPS ≥, 20 female), aged 21.85–77.77 years (mean age 47.41 ± 11.92 years; median 47.88 years) were treated with TTFields for ndGBM, and compared to 52 control patients (15 females); aged 27.03–76.67 years (mean age 51.38 ± 12.55 years; median 51.74 years. All patients from both groups underwent gross total or partial resection of GBM. When available, MGMT promoter methylation status was comparable in both groups. The most frequent AE from TTFields therapy was skin irritation at the array site that usually responded to local corticosteroid application. Patients on TTFields therapy demonstrated improvements in PFS and OS relative to controls (hazard ratio: 0.55, p=0.006; and 0.61, p=0.027 respectively). TTFields average time on therapy was 74.8% (median 82%): median PFS of these patients was 18.16 months. Seven patients with TTFields usage ≤ 60% (23–60%, mean 46.3%, median 53%) had a median PFS of 7.95 months (p=0.0356). Control patients with no TTFields exposure had a median PFS of 11.04 months. In conclusion, this is one of the most extensive studies of ndGBM patients treated with TTFields, covering a period of 18 years in a single center. We demonstrate positive effects of TTFields on both PFS and OS in patients with ndGBM.
INNV-13. INDOLENT H3 K27-ALTERED DIFFUSE MIDLINE GLIOMA
Authors: Damodharan Sudarshawn , Helgager Jeffrey , Puccetti Diane
Published: November 2022

INTRODUCTION

H3 K27-altered diffuse midline glioma (DMG) is an aggressive central nervous system tumor that is universally fatal with a median survival of 8-12 months after diagnosis. Here we present a patient who was incidentally found to have a lesion within the right thalamus on brain magnetic resonance imaging (MRI). Twelve years later, she was found to have a brain mass within the same area with pathology consistent with an H3 K27-altered DMG. Case Description: A 14-year-old female presented with new onset left sided numbness and weakness, blurry vision and a right sided temporal headache. Her past medical history is significant for severe persistent asthma and anoxic brain injury secondary to a cardiopulmonary arrest at 2-years-of-age due to an asthma exacerbation. She has had multiple MRIs of her brain since her initial insult which initially showed the presence of a T2/FLAIR hyperintense lesion within the right medial thalamus in addition to chronic central nervous system changes. The right thalamic lesion was stable in size between images obtained at 2-years and 4-years of age. Her current physical exam is significant for decreased strength to her left upper and lower extremities. Brain MRI with and without contrast is obtained, demonstrating partial effacement of the third ventricle due to mass effect from an enlarging mass from the right thalamus. Biopsy of the lesion demonstrated an H3 K27-altered DMG, WHO grade 4. Our patient went on to receive palliative radiation therapy with 59.4 Gy over 33 fractions.

DISCUSSION

This case illustrates an unusual presentation of an H3 K27-altered DMG with an indolent course, diagnosed twelve years after the initial MRI finding. This proposes the possibility of multiple factors playing a role in the oncogenesis of these aggressive tumors and that further research is warranted.
INNV-18. PROLONGED REMISSION AFTER TREATMENT WITH SINGLE-AGENT IBRUTINIB AS MAINTENANCE THERAPY FOLLOWING SALVAGE RADIATION THERAPY FOR REFRACTORY/RELAPSE PRIMARY CNS LYMPHOMA
Authors: Du Steven , Bota Daniela , Kong Xiao-Tang
Published: November 2022

INTRODUCTION

Primary central nervous system lymphoma (PCNSL) is a rare and aggressive disease. Standard induction therapy for PCNSL consists of a high-dose methotrexate-based (HD-MTX) regimen. For relapsed or refractory primary CNS lymphoma (r/r PCNSL), most of the non-standard treatment regimens including additional chemotherapy, stem cell transplant, or whole brain radiation therapy (WBRT) may not reach long-term remission due to intolerance of the treatment by the patients or lack of effects. Here we report a case with r/r PCNSL reached more than 50-month remission after salvage WBRT followed by single agent ibrutinib maintenance therapy. CASE REPORT: a 75-year-old female with PCNSL, experienced disease recurrence, despite undergoing multiple lines of treatment including initial HD-MTX, second-line rituximab and temozolomide, repeated HD-MTX, and a clinical trial. Because her disease was refractory to these treatment options, we decided to start WBRT as salvage therapy, which resulted in significant reduction of tumor burden. Prior studies, however, showed that r/r PCNSL patients who underwent salvage WBRT alone had a median survival of only 1-2 years. Therefore, to extend the remission after salvage WBRT, she opted to try ibrutinib, a Bruton’s tyrosine kinase inhibitor with past success in treating r/r PCNSL patients, as maintenance therapy after salvage WBRT. She tolerated well and decided to be off the treatment after 18 month-maintenance therapy. The patient has been stable clinically. She has since survived for more than 50 months after the completion of salvage WBRT plus maintenance therapy with ibrutinib.

DISCUSSION

Compared to prior reports of 11 to 16 months of median survival after salvage WBRT alone for r/r PCNSL, our case has had prolonged survival, which supports the potential positive effect of ibrutinib as maintenance therapy for r/r PCNSL after salvage WBRT