bluuebunny/crossref_metadata_2025_split huggingface dataset:
IMMU-03. UTILIZING IN SILICO SINGLE-CELL TRANSCRIPTOMIC AND TCR PROFILING FOR THE IDENTIFICATION AND ISOLATION OF TUMOR-REACTIVE TCR CLONOTYPES IN RECURRENT GLIOBLASTOMA PATIENTS
Authors: Lee Alexander , Sun Lu , Chow Frances , Treger Janet , Cook Danielle , Porter Ely , Chen Xi , Herrera Gilbert , Everson Richard , Liau Linda , Cloughesy Timothy , Hugo Willy , Nathanson David , Prins Robert 
Published: November 2023

Abstract

BACKGROUND
Glioblastoma (GBM) is the most aggressive form of primary brain cancer with no current cure. Immunotherapies, such as checkpoint blockade and adoptive T cell therapy, have effectively treated other cancers, but in GBM, these therapies have shown limited efficacy. This may be partially due to a lack of infiltrating tumor-reactive T cell populations capable of eliminating the tumor.

METHODS
&

RESULTS
We used single-cell RNA and TCRsequencing to examine the transcriptome and TCR repertoire of tumor-infiltrating T cells from a cohort of 32 recurrent GBM patients, 18 of whom had received neoadjuvant anti-PD-1 therapy. After dimensional reduction, we found that the cluster defined by T cell activation and exhaustion gene expression had the greatest number of expanded T cell clones and a transcriptomic profile similar to a previously published signature of tumor-reactive T cells. Focusing on a patient treated with neoadjuvant anti-PD-1 and a EGFRvIII Bispecific T cell engager therapy, we selected TCRs from T cells within the activated and exhausted cluster. Utilizing the PathFinder DNA Assembly technology developed by Rootpath, we generated a TCR library containing the selected TCRs and transduced the library into a fluorescent-reporter Jurkat line that measures T cell activation. We co-cultured the transduced Jurkats with the patient-derived gliomasphere, FACs sorted for activated Jurkats and performed TCR sequencing on the sorted cells. Six TCRs within the sorted population were over-represented relative to sorted T cells from control co-cultures, indicating their tumor reactivity.

CONCLUSIONS
In this study we provide a proof-of-concept framework for how incorporating silico single-cell analysis of a T cell’s transcriptome and TCR can be used to identity tumor-reactive T cells for potential downstream adoptive T cell therapies for patients with recurrent GBM.
IMMU-11. NLRP3+ TUMOR-ASSOCIATED MYELOID CELLS ARE ENRICHED IN ASTROCYTIC GLIOMAS AND REPRESENT A THERAPEUTIC VULNERABILITY
Authors: Chandramohan Vidya , Stevenson Kevin , Yu Yen-Rei Andrea , Reitman Zachary , He Yiping , McLendon Roger , Osorio Cristina , Gregory Simon , Lipp Eric , Desjardins Annick , Friedman Allan , Nair Smita , Patel Anoop , Bigner Darell , Ashley David 
Published: November 2023

Abstract

BACKGROUND
Tumor-associated myeloid cells (TAMs) are known drivers of immunosuppression and glioma progression. Hence novel approaches for repolarizing TAMs from a protumorigenic to an antitumorigenic phenotype is an active area of investigation. While single-cell RNA sequencing (scRNA-seq) studies have characterized glioma TAMs in great detail, their uniqueness or similarity to TAMs in other tumors remains unknown. Cross-comparison of glioma TAMs with TAM subsets from other tumor types will aid in identifying novel TAM-targeted immunotherapies.

METHODS
We performed scRNA-seq of ~26K myeloid cells from nine patients with high-grade astrocytoma (n=3) and oligodendroglioma (n=6). The scRNA-seq findings were validated using TCGA RNA-seq data, public scRNA-Seq data, and immunohistochemistry (IHC) analysis.

RESULTS
The TAM clusters in gliomas predominantly exhibited a microglia-specific gene signature, while macrophage and monocyte clusters were also identified. Utilizing canonical TAM markers, we identified three major TAM subtypes, SPP1+ regulatory, FOLR2+ tissue-resident, and NLRP3+ inflammatory, enriched in astrocytomas. A gene set enrichment analysis of the three TAM subtype genes confirmed their enrichment in astrocytomas (IDHMut-non-codel) (P=0.0005; normalized enrichment score [NES]=1.79) in the TCGA bulk RNAseq data from IDH1-mutant LGGs. Investigation of the three TAM subtype markers in public scRNA-seq data confirmed their enrichment in immune/myeloid clusters across multiple glioma types (gangliogliomas, pilocytic astrocytomas, astrocytomas, diffuse midline gliomas, and glioblastomas). Finally, IHC analysis of 14 astrocytomas demonstrated regional variation in NLRP3 and FOLR2 expression in all cases, with a trend toward an inverse association between NLRP3 and FOLR2 expression.

CONCLUSIONS
1) Astrocytomas (IDHMut-non-codel) comprise TAM subsets common across multiple tumor types. 2) The FOLR2+ TAMs predict favorable outcomes, while the NLRP3+ TAMs are immunosuppressive and predict adverse outcomes in breast cancer. Given the inverse expression of NLRP3 and FOLR2 in astrocytomas, we hypothesize that NLRP3 TAMs+ are immunosuppressive and a potential immunotherapeutic target in astrocytomas.
IMMU-15. INTERPLAY BETWEEN ATRXAND IDH1 MUTATIONS GOVERNS INNATE IMMUNE RESPONSES IN DIFFUSE GLIOMAS
Authors: Whitfield Benjamin , Hariharan Seethalakshmi , Pirozzi Christopher , Waitkus Matthew , Brown Michael , Bowie Michelle , Irvin David , Roso Kristen , Fuller Rebecca , Hostettler Janell , Dharmaiah Sharvari , Gibson Emiley , Briley Aaron , Mangoli Avani , Fraley Casey , Shobande Mariah , Stevenson Kevin , Zhang Gao , Malgulwar Prit Benny , Roberts Hannah , Roskoski Martin , Spasojevic Ivan , Keir Steve , He Yiping , Castro Maria , Huse Jason , Ashley David 
Published: November 2023

Abstract
Stimulating the innate immune system has been explored as a therapeutic option for the treatment of gliomas. Inactivating mutations in alpha-thalassemia retardation X-linked (ATRX) are defining molecular alterations in IDH-mutant astrocytomas and have been implicated in dysfunctional immune signaling. However, little is known about the interplay between ATRX loss and IDH mutation on innate immunity. To explore this interaction, we used both CRISPR and the RCAS/tva system to generate murine models of ATRX-deficient glioma, in the presence and absence of the IDH1 R132H mutation. We found that ATRX-deficient glioma cells were exquisitely sensitive to dsRNA-based innate immune agonism, displaying increases in inflammatory gene expression and profound cytokine release in vitro, while mice bearing ATRX KO tumors displayed increased T-cell infiltration and improved survival. The presence of IDH1 R132H dampened baseline expression of key innate immune genes and cytokines, with genetic and pharmacological inhibition of IDH1 R132H partially rescuing this suppression. Despite the suppression observed at baseline, IDH1 R132H expression did not interfere with the ATRX KO-mediated sensitivity to dsRNA agonism. These results demonstrate that ATRX loss primes cells for recognition of and activation against dsRNA, while IDH1 R132H reversibly masks this priming. Thus, this study suggests that innate immunity is a potential therapeutic vulnerability of astrocytoma. Further studies are in progress to validate the influence of ATRX loss on immune signaling and tumor-immune coevolution within human tumors.
IMMU-17. INDUCING IMMUNE RESPONSE WITH FLASH AND CONVENTIONAL DOSE-RATE RADIATION IN DIFFUSE MIDLINE GLIOMA (DMG)
Authors: Gartrell Robyn , Minns Hanna , Padilla Oscar , Wei Hong-Jian , Webster-Carrion Andrea , Tazhibi Masih , McQuillan Nicholas , Zhang Xu , Gallitto Matthew , Yeh Rebecca , Zhang Zhiguo , Szalontay Luca , Pavisic Jovana , Garty Guy , Garvin James , Canoll Peter , Vanpouille-Box Claire , Menon Vilas , Olah Marta , Rabadan Raul , Wu Cheng-Chia 
Published: November 2023

Abstract
Diffuse Midline Glioma is a universally fatal inoperable pediatric brain tumor associated with H3K27M mutation and median survival of 9-15 months. Radiation therapy (RT) is the primary treatment, extending life by only three months. Conventional dose-rate RT (2Gy/min, CONV) can induce an immune response with type 1 interferon (IFN1) in multiple cancers including adult brain tumors. Ultra-fast dose-rate RT (90Gy/second, FLASH) has comparable tumor control to CONV with decreased toxicity, however, evaluation of immune response is limited. Using an orthotopic syngeneic model of brainstem DMG, we performed single-cell RNA sequencing (scRNA-seq) on CD45+ immune cells isolated from tumors four days post 15Gy FLASH or CONV RT, and compared to unirradiated tumor. We performed flow cytometry at day 4 and day 10 post-RT. ScRNA-seq reveals 33,308 immune cells in 17 unique clusters. Microglia (MG) make up 73.8% and non-resident myeloid cells make up 7.9% of cells and include macrophages (MAC), dendritic cells (DC) and Monocytes (MONO). Flow at day 4 and day 10 show no significant difference in proportion of immune cell subtypes comparing FLASH, CONV and tumor. With differential gene expression, FLASH and CONV provoke a similar inflammatory and IFN1 response in MG while in MACs and DCs IFN1 pathway expression is increased in CONV compared to FLASH (p&lt; 0.001 for both), confirmed by flow at day 4 using IFNAR1 expression (MACs p&lt; 0.05, DCs p&lt; 0.01). Flow at day 10 post-RT shows increased IFNAR1 expression in MACs in both CONV and FLASH compared to tumor (p&lt; 0.0001) while day 10 IFNAR1 in DCs is similar in all three groups. In summary, while immune proportions are similar comparing different dose rates of RT, IFN1 is differentially expressed in CONV compared to FLASH at day 4 in MACs and DCs, while this response at day 10 is similar comparing dose rates.
IMMU-19. MUTANT ISOCITRATE DEHYDROGENASE 1 IN GLIOMA CAUSES A REDUCTION IN ADENOSINERGIC PATHWAY SIGNALING WITHIN THE TUMOR MICROENVIRONMENT
Authors: McClellan Brandon , Dabaja Ali , Banerjee Kaushik , Zhu Ziwen , Barissi Marcus , Oh Paul , Wei Abraham , Lowenstein Pedro , Castro Maria 
Published: November 2023

Abstract
Glioma is one of the most aggressive cancers and represents approximately 80% of malignant brain tumors. The majority of CNS World Health Organization (WHO) grades 2-3 gliomas and a subset of high-grade gliomas (CNS WHO grade 4) harbor mutated isocitrate dehydrogenase 1 (IDH1R132H; mIDH1). Mutant IDH1 results in the accumulation of 2-hydroxyglutarate (2HG) which elicits profound changes in the glioma transcriptome/biology. Consequently, mIDH1 glioma patients have a significantly increased median survival compared to wildtype IDH1 patients. Recent data from our lab showed that mIDH1 gliomas exhibit increased immune-reactivity of tumor-infiltrating immune cells. A critical pathway that mediates reduction of anti-tumor immune cell reactivity is the adenosinergic pathway (AP), which converts ATP into adenosine via the enzymes CD39 and CD73. Adenosine binds to adenosine receptors, A2AR and A2BR, on immune cells resulting in a decreased immunoreactivity. Although mIDH1 has been implicated in altering the AP, the extent to which the AP is affected by mIDH1 in gliomas is largely unknown. Here we used human and mouse mIDH1 glioma cells in vitro along with in vivo mIDH1 glioma mouse models to examine changes in the AP. Our results show reduced levels of the enzyme, CD73, on the surface of mIDH1 glioma cells, and the ATP concentration in the mIDH1 glioma cell growth media is lower than from wildtype IDH1 glioma cells. Additionally, we discovered that mIDH1 glioma-infiltrating macrophages have decreased expression of CD39 and A2AR compared to their wildtype IDH1 glioma counterparts. Taken together, these data suggest a reduction of the AP mediated-immune suppression in mIDH1 gliomas. Uncovering these differences in the AP provides novel insights on the use of AP inhibitors for glioma treatment, revealing whether the AP is a preferred target in specific glioma types based on the IDH1 mutation status.
IMMU-20. MESSENGER RNA CHALLENGE RAPIDLY PREDICTS BRAIN TUMOR RESPONSIVENESS TO CHECKPOINT INHIBITORS
Authors: Castillo Paul , Ogando-Rivas Elizabeth , Geffrard Hillary , Pepe Alfonso , Liu Ruixuan , Nguyen Duy , Pedro Diego , Zhang Dingpeng , DeVries Anna , Qdaisat Sadeem , Grippin Adam , Karachi Aida , Rahman Maryam , Milner Rowan , Huang Jianping , Ligon John , Silver Natalie , Li Derek , Lee Ji-Hyun , Carrera-Justiz Sheila , Weidert Frances , Mitchell Duane , Mendez-Gomez Hector , Sawyer W Gregory , Sayour Elias 
Published: November 2023

Abstract

BACKGROUND
Pathogen recognition receptor (PRR) activation is requisite for igniting the immune response and making the tumor microenvironment ‘hot’ during oncogenesis. We theorized that these PRRs would remain present in ICI responsive tumors and could be challenged using pan-PRR mimic to prospectively predict brain cancer immunogenicity and response to immune checkpoint inhibitors (ICIs).

METHODS
By layering mRNA into multilamellar nanoparticles (ML RNA-NPs), multiple pathogen recognition receptors can be simultaneously activated in individualized cancer cells serving as a broad screen to test tumor immunogenicity. We challenged murine brain tumor lines known to respond (GL261 and SMA-560) or resist ICI treatment (KR158b and CT2A) with ML RNA-NPs and analyzed downstream production of proinflammatory cytokines as predictors of ICI response.

RESULTS
We demonstrated that ML-RNA-NPs can activate multiple PRRs simultaneously. Following ML RNA-NP challenge, ICI responsive tumors GL261 and SMA-560 (compared to more ICI resistant lines KR158b and CT2A) elicited increased production in pro-inflammatory cytokines such as IFN-β and IL-6 and in CCL4. To further develop this work into a commercially viable tool to predict ICI response, we developed 3D modeling of glial tumors for ML RNA-NP challenge and immunogenicity prediction. We enrolled canines with primary gliomas and began growing their tumoroids in 3D using liquid-like solid (LLS) technology. Using this pipeline, we successfully perfused 3D tumoroids with ML RNA-NP demonstrating ability to set up real-time patient derived explants to predict immunogenicity. We demonstrated that mRNA perfusion of 3D tumoroids elicited similar cytokine response observed with 2D culture of cell-lines.

CONCLUSION
Our results indicate that cytokine signatures following ML RNA-NP challenge differ significantly from ICI responsive versus non-responsive brain tumors. These findings allow for creation of a diagnostic assay to quickly assess brain tumor immunogenicity, allowing for informed treatment with ICIs.
IMMU-27. NON-TUMOR CELL IDO1 NON-ENZYMATICALLY DECREASES RT + PD-1 MAB TREATMENT EFFICACY IN OLDER ADULTS WITH GLIOBLASTOMA
Authors: Zhai Lijie , Bell April , Asimakidou Evridiki , Lauing Kristen , Kim Miri , Bommi Prashant , Barton Kevin , Borys Ewa , Anderson Douglas , Prabhu Vikram , Walunas Theresa , Schiltz Gary , Allen Jacob , McCusker Robert , Thakkar Jigisha , Lukas Rimas , Wainwright Derek 
Published: November 2023

Abstract
Checkmate 498 is a large, randomized, phase 3 clinical trial that demonstrated an improved hazard ratio for younger but not older adults with MGMT unmethylated GBM treated with RT + PD-1 mAb. In an effort to better understand the biological determinant, indoleamine 2,3 dioxygenase 1 (IDO1), and its relationship with reducing RT + PD-1 mAb treatment efficacy in older adults with GBM, we created and/or compared 20-26 month-old C57BL/6 background mice that were IDO1-T2A-GFP [wild-type (WT); negative control (n=16)], IDO1 enzyme null [with normal IDO1 expression potential but incapable of tryptophan metabolism (n=16)], IDO1KO (n=4), Ido1fl/fl (n=14), Lysm-Cre+→Ido1fl/fl [IDO1-deficient in monocytes, macrophages, and microglia (n=20)], CD11c-Cre+→Ido1fl/fl [IDO1-deficient in dendritic cells (n=23)], Tie2-Cre+→Ido1fl/fl [IDO1-deficient in endothelial cells (n=14)]. Mice were intracranially (ic.) engrafted with 5×103 syngeneic GL261 followed by treatment with radiation (RT) + PD-1 mAb beginning at 14-days post ic. IDO1 WT and IDO1 enzyme null mice had a survival rate of 12.5% and 18.75% at 90-day post ic., respectively, which was significantly reduced as compared to IDO1KO mice with a 75% survival rate (p&lt; 0.05). Tie2-Cre+→Ido1fl/fl mice, CD11c-Cre+→Ido1fl/fl, and Lysm-Cre+→Ido1fl/fl mice had a 100%, 96%, and 70% survival rate at 90-day post ic., respectively, as compared to Ido1fl/fl mice with a 42% survival rate (p&lt; 0.05). Significantly reduced kynurenine (KYN) levels were found in IDO1 enzyme null and IDO1KO mice as compared to WT mice (p&lt; 0.001). Unexpectedly, kynurenine levels were also reduced in Tie2-Cre+→Ido1fl/fl but not in CD11c-Cre+→Ido1fl/fl, Lysm-Cre+→Ido1fl/fl or Ido1fl/fl mice (p&lt; 0.05). Tryptophan (TRP) levels did not change between mouse groups. These data support the hypothesis that non-tumor cell IDO1 reduces immunotherapeutic efficacy in older adults with GBM through a TRP-KYN pathway-independent mechanism.
IMMU-31. B-CELL THERAPY (BVAX) PROMOTES CD8 T CELLS' PERSISTENCE AND FUNCTION IN THE GLIOBLASTOMA MICROENVIRONMENT BY MAINTAINING THEIR STEM-LIKE FEATURES
Authors: Wan Hanxiao , Hou David , Katz Joshua , Wang Si , Castro Brandyn , Chia Tzu-Yi , Cervantes Gustavo Ignacio Vazquez , Billingham Leah , Arrieta Victor , Han Yu , Lopez-Rosas Aurora , Zhang Peng , Miska Jason , Lee-Chang Catalina 
Published: November 2023

Abstract
The last decade witnessed tremendous improvement in immunotherapy to treat cancers. Its application in GBM has been particularly challenging due to the immunosuppressive tumor microenvironment, which greatly challenges T-cell infiltration and function. We developed a B-cell therapy with potent antigen-presenting functions that promote CD8+ tumor tropism and persistence in the tumor microenvironment. The present study investigates how BVax activates CD8+ T cells differently from other professional APCs like dendritic cells (DC) that could explain the persistence of CD8+ T cells in the tumor microenvironment. Upon in vitro activation by coculturing with BVax, most CD8+ T cells proliferated into TCF+ PD1- intermediate state, with CD44+ CD62L+ memory phenotype. Less than 10% of CD8+ were in the terminally-proliferation state. In contrast, DCs preferentially expanded TCF- PD1+ terminally-proliferating CD8+ T cells that were exhibiting CD44+ CD62L- effector phenotype. In vivo, activation of CD8+ T cells by co-injecting BVax or DC with CD8+ T cells to CT2A-bearing mice indicated that BVax promoted the proliferation of tumor-reactive CD8 T cells while maintaining the low PD1+ expression. Re-exposing the ex vivo-activated CD8+ T cells to tumor by adoptive transferring them to CT2A tumor-bearing mice revealed that BVax elicited immunological memory and potentiated GzmB+ production of CD8+ T cells. This study suggests that BVax and DC have distinctive interactions with CD8+ T cells, with the former poise CD8+ T cells at a more stem-like state, allowing them to differentiate upon tumor encounter.
IMMU-32. CAN PEMBROLIZUMAB TREAT RECURRENT GLIOBLASTOMA AND REVERSE HIV LATENCY?
Authors: Kaur Jaspreet , Silveira William , MacDonald Robert Loch , Mo Fan , Bao Silin , Yuen Carlen 
Published: November 2023

Abstract
Immunotherapy revolutionized treatment for many solid tumors. Early studies show that immunotherapy may be effective for recurrent glioblastoma. The ongoing Phase 2 LEEP-005 clinical trial (pembrolizumab+lenvima) is currently enrolling glioblastoma patients, but excludes patients with HIV. However, there is a strong need to treat this special patient population. One of the barriers to achieving cure in HIV patients is HIV latency within infect CD4+ T cells. Recently published data suggests that pembrolizumab can reverse HIV latency by forcing the HIV out of intracellular hiding into the plasma where anti-retroviral therapy can be effective. We present a case of a 39 year-old male with HIV and a Left Parietal Glioblastoma who had progressive disease following a subtotal resection and radiation alone due persistent neutropenia. Post-radiation MR brain wwo showed progressive disease in the left parietal lobe and new dural-based lesions in the left frontal and temporal lobes. HIV viral load was &lt; 20 and CD4 count was 406 on HAART. The patient declined lenvima, but was amenable to bevacizumab. At 1 month follow-up, after 1 infusion of pembrolizumab 200mg and 2 infusions of bevacizumab, he achieved CR in the original site of disease and stable disease in the left frontal and temporal lobes. HIV viral load was 52. CD4 count was 660. Though it is too early to draw any firm conclusions, in our patient, pembrolizumab appears to induce HIV reversal, which is supported by an increase in the increase in his HIV viral load. The combination of pembrolizumab and bevacizumab appears to be effective at short-term follow-up. Our study is significant because there is limited evidence on the use of PD-L1 inhibitors in active HIV patients with cancer. To our knowledge, our case is the first reported case of the use of pembrolizumab in a patient with HIV and glioblastoma.
IMMU-37. TARGETING NON-CATALYTIC ACTIVATORS OF THE PROTEASOME DECREASES TUMOR GROWTH AND ENHANCES ANTIGEN PRESENTATION IN GLIOBLASTOMA
Authors: Heemskerk Kyle , Hao Xiaoguang , Cseh Orsolya , Luchman H Artee , Weiss Samuel 
Published: November 2023

Abstract
Glioblastoma (GBM) is the most common adult primary brain tumor plagued by inevitable recurrence and poor survival. We recently performed a genome wide CRISPR/Cas9 essentiality screen in GBM stem cells, revealing a plethora of potential targets for further exploration. The proteasome is a multimeric protein complex that degrades cellular proteins contributing to homeostatic proteostasis, stress response, and antigen presentation. Most proteasomal subunits are essential for GBM stem cell growth in vitro, however, they are also essential for non-malignant neural cells, suggesting that inhibition of those subunits may lead to toxicity. Indeed, adverse neurological symptoms were prevalent in phase III clinical trials for the brain penetrant proteasome inhibitor, Marizomib, which may be linked to the vital role of proteasome subunits in non-malignant neural counterparts. Proteasome inhibitors target the catalytic subunits of the proteasome, however the role of individual proteasome activators, most of which are non-essential for growth in vitro, have not been fully elucidated in GBM. Here, we examined the functionality of non-essential proteasome activator subunits in GBM stem cells in vitro and in vivo. Surprisingly, despite lack of growth changes in vitro, we observed abrogated stem-cell self-renewal in vitro and improved survival in vivo in orthotopic xenograft models following targeting of specific activator subunits. Molecular profiling of targeted cells revealed an upregulation of interferon-γ signaling and upregulation of antigen presentation machinery. Thus, targeting specific activator subunits may inhibit malignant growth in vivo while sparing normal neural counterparts from proteotoxic stress. We are further investigating enhanced antigen presentation by targeting these proteasome activator subunits and examining changes in the tumor microenvironment and survival in syngeneic immunocompetent models of GBM. Further understanding of this mechanism may provide novel targets for GBM treatment or improve immunotherapies in GBM.