Immune Phenotype Research Articles

Abstract B036: WEE1 confers resistance to PD-1 blockade via hyperactivation of the AKT signaling pathway

Abstract Immune checkpoint blockade (ICB) has revolutionized cancer treatment, but resistance limits its clinical effectiveness. Therefore, understanding immune resistance mechanisms and identifying predictive markers are essential. Our transcriptomic analysis of PD-1 blockade-treated patients and non-responsive tumor models identifies WEE1 as a key resistance factor, promoting cancer stem cell (CSC)-like traits and immune resistance phenotypes including low T cell infiltrated into tumor and anti-apoptotic properties to cytotoxic T cells (CTLs). WEE1 drives AKT hyperactivation, enhancing expression of resistance factors such as CYCLIN A which contributes to CSC-like properties, and the anti-apoptotic molecule MCL-1, while also reducing T cell infiltration via CXCL10 downregulation. This signaling axis is conserved across various cancers. Notably, WEE1 inhibition sensitizes tumors to ICB by disrupting these refractory properties and reinvigorating antitumor immunity. These findings underscore the novel role of WEE1 in driving immune resistance and CSC-like characteristics, supporting the use of WEE1 inhibitors as a strategy to overcome resistance to anti-PD-1 therapy. Citation Format: Hyo-Jung Lee, Suyeon Kim, Eunho Cho, Tae Woo Kim. WEE1 confers resistance to PD-1 blockade via hyperactivation of the AKT signaling pathway [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B036.

The lung is a megakaryocyte outpost that can defend against thrombocytopenic attack.

Lung megakaryocytes (Mks) are a unique subset of Mks that are distinct from their bone marrow counterparts. Recent evidence suggests that lung Mks favor an immune phenotype, but have unclear contributions to the total platelet mass. In this issue of the JCI, Livada et al. used an array of complementary in vivo labeling and tracing models in mice to investigate a longstanding question of where lung Mks are derived. By combining these models with stressed conditions, the authors assessed the contribution of lung Mks to total platelet counts in a homeostatic and thrombocytopenic state. Mks were minor contributors to the circulating pool of platelets during homeostasis but increased output during thrombocytopenia. These findings add critical understanding to the development of lung Mks and demonstrate the dynamic potential of these specialized cells to respond to thrombocytopenia.

Gadoxetic Acid-Enhanced Magnetic Resonance Imaging Features Can Predict Immune-Excluded Phenotype of Hepatocellular Carcinoma

Introduction: Immunotherapy is the first-line treatment for intermediate-advanced stage hepatocellular carcinoma (HCC), although its outcomes vary. This study aimed to identify imaging biomarkers of immunotherapy susceptibility linked to gadoxetic acid-enhanced magnetic resonance imaging (EOB-MRI) and immune phenotypes, particularly immune-excluded phenotypes, with a tumor immune barrier. Methods: We performed immunohistochemical staining with a CD8+ antibody, and samples were classified into immune-inflamed, -intermediate, -excluded, and -ignored phenotypes. We assessed EOB-MRI findings obtained from 104 patients who underwent hepatectomy for HCC and evaluated the relationship between MRI findings and immune phenotype. Spatial transcriptome analysis of tumor tissues in each immune phenotype was performed to characterize the MRI findings. For validation, we analyzed the treatment effect on 60 nodules in another cohort of 27 patients who received combined immunotherapy using anti-programmed death-ligand 1 and anti-vascular endothelial growth factor (VEGF) antibodies. Results: HCCs with rim arterial phase hyperenhancement (APHE) (odds ratio [OR] 17.3 P=0.009), peritumoral enhancement on the arterial phase (OR 8.6, P<0.004), and intermediate intensity on the hepatobiliary phase (HBP) measured with a visual 3-point scale (OR 28.2, P=0.002) were associated with immune-excluded phenotype, where tumors tended to be larger and of the single nodular type with extranodular growth and confluent multinodular rather than the simple nodular type. Spatial transcriptome analysis revealed a spatial relationship among cytotoxic T lymphocytes, VEGF signals, and cancer-associated fibroblasts at the tumor-invasive margins in this phenotype. From the validation study, nodules with any one of these three imaging findings had a significantly prolonged time to-nodular progression (P=0.007, median not reached vs. 226 days). Conclusion: HCCs with rim APHE, peritumoral enhancement on arterial phase, and intermediate intensity on HBP with visual 3-point scale could be non-invasive biomarkers to predict the immune-excluded phenotype with tumor immune barrier. These HCCs were most likely to respond to the combined immunotherapy.

Evaluating the causal association between circulating plasma proteins, 731 immune cell phenotypes, and atopic dermatitis: A mediation Mendelian randomization study.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by eczematous lesions and severe itching. However, its pathogenesis has not yet been fully elucidated. The aim of this study was to investigate the causal relationship between plasma proteins and AD, as well as to identify and quantify the potential roles of immune cell phenotypes as mediators. We utilized summary-level data from genome-wide association studies and conducted a two-sample Mendelian randomization (MR) analysis involving 4907 circulating plasma proteins, 731 immune cell phenotypes, and AD. Initially, we conducted bidirectional univariate MR analyses to forecast causal effects linking circulating plasma proteins and AD. Subsequently, we employed a two-step MR analysis to scrutinize the immune cell phenotypes that could mediate these effects. The inverse variance weighted was the main method employed for MR analysis, while the Cochran's Q test and MR-Egger intercept test were used to assess the presence of heterogeneity and pleiotropy, respectively. We then determined whether our results could be influenced by individual single-nucleotide polymorphisms using the "leave-one-out" test. Positive correlations were observed between KRT1, IL18R1, and SEMA6A and the risk of AD, whereas BDH2, ADAMTS3, ANKRD1, TIAM1, MID2, and IFNA16 all showed negative correlations with the risk of AD. Mediation analysis indicated that CD8 on CM CD8br cells acted as a mediator between IFNA16 and AD, with a mediation effect proportion of 11.2%. In addition, sensitivity analyses did not reveal significant heterogeneity or level pleiotropy. Our findings indicated the presence of a one-way causal relationship between the circulating plasma protein IFNA16 and AD. This study also explored immune cell phenotypes that may serve as mediators, offering novel insights into the etiology, pathogenesis, and potential clinical interventions in AD. Nevertheless, these findings need to be validated by clinical and laboratory studies.

Association between immune cells and urticaria: a bidirectional Mendelian randomization study

Urticaria is characterized by transient itchy symptoms on the skin, usually accompanied by swelling, which is caused by mast cell activation leading to increased vascular permeability and dilation of the dermis. Urticaria involves recurrent activation of mast cells, T cells, eosinophils, and other immune cells around lesioned venules, with complex regulatory systems affecting mast cell functions, potentially contributing to urticaria pathogenesis. The direct causal relationship between immune cells and urticaria is currently unclear. To address this, our study utilized a bidirectional Mendelian randomization analysis, employing instrumental variables (IVs) associated with immune cells and urticaria, to investigate this causal relationship. First, by utilizing Genome-wide Association Study (GWAS) data, we identified 31 immunophenotypes associated with urticaria risk, with 18 increasing and 13 decreasing the risk. Through rigorous criteria, we identified 4 immunophenotypes that have a strong causal relationship with urticaria. Notably, HLA DR+ CD4+AC, CD45 on CD8br, and HLA DR on plasmacytoid dendritic cells were associated with an increased risk, while CD8dim NKT %lymphocyte was identified as a protective factor. Sensitivity analyses, including the MR-Egger intercept test, scatter plots, funnel plots, and leave-one-out analysis, supported the robustness of the findings. Reverse MR analysis suggested an inverse causal effect of urticaria on CD8dim NKT %lymphocyte, reinforcing the potential bidirectional nature of the relationship between urticaria and immune cell phenotypes. Our research substantiates the bidirectional causal relationship between immune cells and urticaria, thus benefiting for urticaria-targeted therapy development.

Vaccination Against Androgen Receptor Splice Variants to Immunologically Target Prostate Cancer

Background/Objectives: Androgen receptor (AR) expression and signaling are critical for the progression of prostate cancer and have been the therapeutic focus of prostate cancer for over 50 years. While a variety of agents have been developed to target this axis, many of these fail due to the emergent expression of AR RNA splice variants, such as AR-V7, that can signal independently of ligand binding. Other therapies, such as vaccination against prostate-specific antigens, have achieved FDA approvals but have fallen short of being incorporated as standard-of-care therapies for advanced prostate cancer. This may be due to the elevated level of immunosuppression observed in prostate cancer, which remains largely refractory to immune checkpoint blockade. Methods: We developed a vaccine targeting AR-V7, a common isoform associated with treatment resistance, and demonstrated its ability to elicit AR-V7-specific immunity and enable anti-tumor responses against AR-V7+ cancers in subcutaneous tumor models. Results: Our studies also revealed that AR-V7 expression conferred an immune suppressive phenotype that was significant in a non-AR-dependent prostate cancer model. Notably, in this model, we found that vaccination in combination with enzalutamide, an AR antagonist, suppressed these aggressive immune suppressive cancers and resulted in enhanced survival in comparison to control vaccinated and enzalutamide-treated mice. While anti-PD-1 immune checkpoint inhibition (ICI) alone slowed tumor growth, the majority of vaccinated mice that received anti-PD-1 therapy showed complete tumor elimination. Conclusions: Collectively, these results validate the importance of AR signaling in prostate cancer immune suppression and suggest the potential of AR-V7-specific vaccines as therapeutic strategies against prostate cancer, offering significant protective and therapeutic anti-tumor responses, even in the presence of androgen signaling inhibitors.

Abstract PR005: Leveraging mass cytometry for phenotyping CTCs in SCLC liquid biopsies: Tracking therapy resistance at a personalized level

Abstract Liquid biopsies present a promising, minimally invasive procedure through which clinicians can assess and monitor disease status by interrogating circulating tumor cell (CTC) and immune cell phenotypes. In the case of Small-cell lung cancer (SCLC) – a type of lung cancer characterized by aggressive growth, dismal prognosis and limited treatment options - leveraging liquid biopsies and CTCs for evaluating disease status would be of great benefit, given that biopsies are scarce. Furthermore, SCLC CTC numbers are higher compared to other cancers, presenting a unique opportunity to utilize them for assessing SCLC therapy resistance and identifying new targetable markers. In this study, we leverage mass cytometry (i.e. CyTOF) to phenotype SCLC CTC phenotypes in longitudinal clinical specimens to assess therapy resistance at a personalized level. By using CyTOF - a multiplex cytometric approach that enables interrogation of 30-50 protein markers per single cell – we show that we are able to identify and analyze a significant number of CTCs and their heterogeneous expression profiles. To achieve this, we first optimized a CyTOF panel of ∼25-30 antibodies by using SCLC cell lines and blood specimens from healthy donors and SCLC patients. We show that by using this approach, we are able to identify and phenotype SCLC CTCs by using the combination of CD45 (negative expression) and CD56 positive expression with further validation achieved by TTF1 and SCLC subtype transcription factor expression (NEUROD1, POU2F3 and ASCL1). This approach avoids enriching for specific CTC epithelial phenotypes that express surface markers that fluctuate during epithelial- mesenchymal transition (EMT) (e.g. EpCAM, a common marker for isolating CTCs). For parallel immune profiling we assessed among others CD8/CD4 (T cells) and CD11b/HLA-DR (Myeloid-derived suppressor cells, MDSCs) expression. We then proceeded to interrogate CTC phenotypic differences in the naïve vs relapsed setting as well as dynamic changes in longitudinal blood specimens from SCLC patients by evaluating markers that describe immune suppression activity (PD-L1) and EMT status (E-Cadherin, Vimentin, MUC1, Twist and Slug). Importantly, we show that with our approach we can track in real time therapeutic targets that are currently being tested in clinical trials or have been recently FDA approved (e.g. pYAP and DLL3) and found that MUC1 and pYAP are significantly increased in CTCs detected in blood specimens of SCLC relapsed patients. Our immune profiling showed an increase of MDSC percentages and a decrease in the CD4/CD8 T cell ratio in the relapsed setting. Finally, when we used PHENOSTAMP, a previously developed reference EMT map for assessing EMT phenotypes in lung cancer clinical specimens, we observed an increase of EMT heterogeneity in CTCs in relapsed patients. Our study highlights the translational power of our approach in utilizing CyTOF for longitudinally tracking CTCs directly in SCLC patient liquid biopsies towards assessing therapy response and resistance at a personalized level. Citation Format: Loukia G Karacosta, Sayantan Bhattacharyya, Allison Stewart, Ashley Victorian, Ester F Lujan, Shafqat Ehsan, Subin Kim, Alberto Duarte, Runsheng Wang, Benedict Anchang, Jing Wang, Lauren Byers. Leveraging mass cytometry for phenotyping CTCs in SCLC liquid biopsies: Tracking therapy resistance at a personalized level [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR005.

Microglia Sing the Prelude of Neuroinflammation-Associated Depression.

Major depressive disorder (MDD) is a psychiatric condition characterized by sadness and anhedonia and is closely linked to chronic low-grade neuroinflammation, which is primarily induced by microglia. Nonetheless, the mechanisms by which microglia elicit depressive symptoms remain uncertain. This review focuses on the mechanism linking microglia and depression encompassing the breakdown of the blood-brain barrier, the hypothalamic-pituitary-adrenal axis, the gut-brain axis, the vagus and sympathetic nervous systems, and the susceptibility influenced by epigenetic modifications on microglia. These pathways may lead to the alterations of microglia in cytokine levels, as well as increased oxidative stress. Simultaneously, many antidepressant treatments can alter the immune phenotype of microglia, while anti-inflammatory treatments can also have antidepressant effects. This framework linking microglia, neuroinflammation, and depression could serve as a reference for targeting microglia to treat depression.

Longitudinal Follow-up Reveals Peripheral Immunological Changes Upon Tick Bite in a-Gal-Sensitized Individuals.

α-Gal syndrome is characterized by specific IgE (sIgE) antibodies to the carbohydrate galactose-α-1,3-galactose (α-Gal) and delayed onset of allergic symptoms after ingestion of mammalian meat. While tick bites are assumed to mediate sensitization, the immune response to tick bites has not yet been investigated. To investigate the peripheral immune response to tick bites in humans over time. In a longitudinal cohort study, immunological reactions associated with tick bites (Ixodes species) were analyzed within 1 day (V1), 2 weeks (V2), 1 month (V3), and 3 months (V4) after the occurrence of a bite. sIgE, sIgG, and sIgG subclass levels, as well as 10 cytokines, were quantified. Deep immune phenotyping was performed using mass cytometry. A total of 4 controls and 10 patients were bitten by a tick and followed up over 3 months. None of the controls developed sIgE to α-Gal, and sIgE increased in all patients from V1 until V2/V3, as did IL-8 levels. We noted a significant increase in CD19+ B cells and B-cell subpopulations, as well as a decrease in γδ CD56+ T cells in patients between V0 and V1. At V1, frequencies of plasmacytoid dendritic cells (pDCs) and γδ CD56+ T cells were lower in patients than in controls. Our study provides evidence of significant changes in several immune cell populations in α-Gal sensitized patients, along with increased levels of IL-8 and sIgE. This is the first exploratory study to investigate longitudinal peripheral immune profiles in patients and controls bitten by ticks.

Causal relationship between immune cells and Guillain-Barré syndrome: a Mendelian randomization study

ObjectiveThe aim of this study was to investigate the causal effect of immune cell phenotype on GBS using two-sample Mendelian randomization (MR) approach.MethodsThis study used MR to investigate the causal relationship between 731 immune cell phenotypes and GBS. We used Inverse variance weighted, Weighted median, MR Egger, Simple mode, Weighted mode for MR analysis. We also used the Cochran Q test, MR-Egger intercept test, IVW regression and MR-PRESSO, leave-one-out analysis to assess the presence of horizontal pleiotropy, heterogeneity and stability, respectively.ResultsOur study revealed a causal relationship between 33 immune cell phenotypes and GBS. Twenty immunophenotypes were observed to be associated with GBS as risk factors. For example, CD20 on IgD+ CD38dim in the B cell group (OR = 1.313, 95%CI:1.042–1.654, p = 0.021), CD3 on CD4 Treg in Treg cell group (OR = 1.395, 95%CI:1.069–1.819, p = 0.014), CD3 on TD CD8br in Maturation stages of T cell group (OR = 1.486, 95%CI:1.025–2.154, p = 0.037), CD16 on CD14+ CD16+ monocyte in Monocyte group (OR = 1.285, 95%CI:1.018–1.621, p = 0.035), CD33dim HLA DR+ CD11b + %CD33dim HLA DR+ in Myeloid cell group (OR = 1.262, 95%CI:1.020–1.561, p = 0.032), HLA DR+ NK AC in TBNK cell group (OR = 1.568, 95%CI:1.100–2.237, p = 0.013). Thirteen immune phenotypes are associated with GBS as protective factors. For example, CD19 on PB/PC in the B cell group (OR = 0.577, 95%CI:0.370–0.902, p = 0.016), CD4 Treg AC in Treg cell group (OR = 0.727, 95%CI:0.538–0.983, p = 0.038), CD11c + monocyte %monocyte in cDC group (OR = 0.704, 95%CI:0.514–0.966, p = 0.030), CX3CR1 on CD14+ CD16− monocyte in Monocyte group (OR = 0.717, 95%CI:0.548–0.939, p = 0.016), Mo MDSC AC in Myeloid cell group (OR = 0.763, 95%CI:0.619–0.939, p = 0.011), CD45 on granulocyte in TBNK group (OR = 0.621, 95%CI:0.391–0.984, p = 0.042).ConclusionThe findings suggest that certain specific immune cell phenotypes, particularly B cell and Treg cell subpopulations, are causally associated with GBS, providing potential targets for the clinical treatment of GBS.

EPCO-46. SPATIAL TRANSCRIPTOMICS ANALYSIS OF GLIOBLASTOMA REVEALS THREE DISTINCT REGIONAL PROGRAMS OF T-CELL INFILTRATION

Abstract Glioblastoma (GBM) resists T-cell killing by developing an immunosuppressive tumor microenvironment. Transcriptomic technologies, such as single-cell RNA sequencing (scRNA-seq), have characterized immune cell phenotypes and putative cell-cell interactions in GBM. However, the tumor microenvironment surrounding T-cells is still not well characterized, motivating the need for spatial analyses. Two publicly-available GBM spatial transcriptomic datasets were analyzed, with one discovery cohort (N=18) and one validation cohort (N=12). Using cell deconvolution with a scRNA-seq reference dataset, areas of high T-cell infiltration were identified and clustered into regional programs based on transcriptomic signatures. To highlight which ligand-receptor interactions are most prominent in these regional programs, a novel spatial enrichment algorithm was applied. The impact of spatially enriched ligand-receptor interactions on overall survival was evaluated using bulk RNA-sequencing data from The Cancer Genome Atlas (TCGA). Three distinct T-cell regional programs (transitional, immunosuppressive, and infiltrative) were found across both the discovery and validation cohort. The infiltrative T-cell regional program resided on the periphery of the tumor with a neural progenitor phenotype (NRGN, VSNL1, SLC17A7). In contrast, the immunosuppressive T-cell region had a higher proportion of malignant cells and a greater mesenchymal tumor signature (ANXA1, CD44, complement pathway). Furthermore, ligand-receptor enrichment analysis showed that the immunosuppressive region had higher tumor and tumor-associated macrophage (TAM) signaling towards CD4 T-cells along the collagen, midkine, and osteopontin pathways. TCGA survival analysis showed that higher collagen and midkine signals were associated with worse overall and disease free survival. Our results identified three distinct regional programs in GBM and highlight a set of signaling pathways associated with T-cell suppression. Our findings suggest that the disruption of these pathways could reduce immunosuppressive signals from tumor and TAMs to T-cells. Subsequent validation with spatial proteomics data in glioblastoma is ongoing.

IMMU-23. NEOADJUVANT ANTI-PD-1 AND ANTI-CTLA-4 COMBINATION THERAPY INDUCED INTRATUMORAL IMMUNOLOGIC ALTERATIONS IN RECURRENT GBM PATIENTS

Abstract Glioblastoma (GBM) is a highly aggressive brain tumor that responds poorly to checkpoint blockade inhibitors (ICIs), such as anti-PD-1. We previously reported that neoadjuvant anti-PD-1 therapy activated and recruited T cells into recurrent GBM (rGBM) tumors, but also increased immune checkpoint interactions between T cells and myeloid cells. To overcome this resistance mechanism, we are currently conducting a phase 1B clinical trial of neoadjuvant anti-PD-1 (Nivolumab, BMS) +/- anti-CTLA-4 (Ipilimumab, BMS) combination therapy for rGBM patients (NCT04606316). To evaluate the local tumor microenvironment effects of these checkpoint inhibitors, we used multiplex immunofluorescence (mIF), bulk RNA sequencing, and T cell receptor sequencing to analyze the intratumoral immune profile of tumors. Using mIF, we are able to determine the density of key immune cell phenotypes within the section of tumor sample as well as the proximity between phenotypes to better understand their interactions. Patients who received the combination therapy (n=15) trended to have a higher median density of cytotoxic T cells (CD45+CD8+; median density of 27.13 cells/mm2 for combination therapy and 13.78 cells/mm2 for anti-PD-1), helper T cells (CD45+CD4+; median density of 33.73 cells/mm2 for combination therapy and 9.82 cells/mm2 for anti-PD-1), monocytes (CD14+), and HLA-DR+ macrophages (CD45+CD14+HLA-DR+) while patients treated with anti-PD-1 alone (n=16) trended to have densities similar to placebo treated patients (n=6). Patients treated with combination therapy are also trending to have a higher density of macrophages in close proximity (≤20μm) to T cells, particularly HLA-DR+ macrophages, suggesting a higher frequency of interactions within their tumor microenvironments. This data set is currently incomplete, but the final data set will be completed by the meeting. Nonetheless, these preliminary results, as well as the bulk RNA sequencing and T cell receptor sequencing, suggest distinct immunologic effects of monotherapy vs. combination neoadjuvant anti-PD-1 and anti-CTLA-4 therapy in the tumor microenvironment of rGBM patients.

STAMBPL1/TRIM21 Balances AXL Stability Impacting Mesenchymal Phenotype and Immune Response in KIRC.

Kidney renal clear cell carcinoma (KIRC) is recognized as an immunogenic tumor, and immunotherapy is incorporated into its treatment landscape for decades. The acquisition of a tumor mesenchymal phenotype through epithelial-to-mesenchymal transition (EMT) is associated with immune evasion and can contribute to immunotherapy resistance. Here, the involvement of STAM Binding Protein Like 1 (STAMBPL1) is reported in the development of mesenchymal and immune evasion phenotypes in KIRC cells. Mechanistically, STAMBPL1 elevated protein abundance and surface accumulation of TAM Receptor AXL through diminishing the TRIM21-mediated K63-linked ubiquitination and subsequent lysosomal degradation of AXL, thereby enhancing the expression of mesenchymal genes while suppressing chemokines CXCL9/10 and HLA/B/C. In addition, STAMBPL1 enhanced PD-L1 transcription via facilitating nuclear translocation of p65, and knockdown (KD) of STAMBPL1 augmented antitumor effects of PD-1 blockade. Furthermore, STAMBPL1 silencing and the tyrosine kinase inhibitor (TKI) sunitinib also exhibited a synergistic effect on the suppression of KIRC. Collectively, targeting the STAMBPL1/TRIM21/AXL axis can decrease mesenchymal phenotype and potentiate anti-tumor efficacy of cancer therapy.

Immune cells mediate the effects of gut microbiota on neuropathic pain: a Mendelian randomization study.

The gut microbiota may be involved in neuropathic pain. However, the causal association between gut microbiota and neuropathic pain remains unclear. Whether immune cells and inflammatory factors mediate the pathway from gut microbiota to neuropathic pain has not been elucidated. We obtained the summary data of 412 gut microbiota, 731 immune cells, 91 inflammatory factors, and five types of neuropathic pain (drug-induced neuropathy, postherpetic neuralgia, sciatica, trigeminal neuralgia, and unspecified neuralgia) from large-scale genome-wide association study (GWAS) datasets and the FinnGen database. We used bidirectional Mendelian randomization (MR) analysis to explore the causal association between gut microbiota and neuropathic pain. Additionally, we conducted a mediation analysis to identify whether immune cells and inflammatory factors act as mediators within these causal relationships. Our study revealed 30 causal relationships between 26 gut bacterial taxa and five types of neuropathic pain, including four associated with drug-induced neuropathy, six with postherpetic neuralgia, five with sciatica, eight with trigeminal neuralgia, and seven with unspecified neuralgia. Moreover, we identified 35 gut bacterial pathway abundances causally involved in neuropathic pain. The reverse MR analysis showed no evidence of reverse causality from gut microbiota to neuropathic pain. Mediation analysis demonstrated that the immune cell phenotype "HLA-DR++ monocyte % leukocyte" mediated the causal relationship between p_Proteobacteria and sciatica with a mediation proportion of 36.15% (P = 0.038), whereas "CD11c on CD62L+ myeloid dendritic cell" mediated the causal pathway from assimilatory sulfate reduction to trigeminal neuralgia with a mediation proportion of 27.90% (P = 0.041). This study identified the causal relationships between several specific gut microbiota and various neuropathic pain subtypes. Additionally, two immune cells may act as potential mediators in the pathways from gut microbiota to neuropathic pain.

IMMU-66. PHYSIOLOGICALLY RELEVANT MURINE GLIOMA MODELS THAT REPRESENT GBM HETEROGENEITY AND ENABLE THE NEXT GENERATION OF DISCOVERY AND THERAPY EFFICACY STUDIES

Abstract To transform cancer drug development pipelines and gain a deeper mechanistic understanding of cancer: immune interactions, we desperately need better experimental models that faithfully recapitulate the cellular heterogeneity, complex and dynamic cell:cell interactions, and therapy responses in humans. To address this need, we developed and characterized a cohort of five new mouse glioma models in the C57BL6/j background. These transplantable tumorsphere models were derived from spontaneous gliomas that formed in genetically engineered mouse models with different oncogenic drivers, including EGFRviii, PDGFA, PTEN, p53, and NF1 mutations. They show a range of responses to standard of care (TMZ+IR) similar to GBM patients and all are resistant, to immune checkpoint inhibitors, anti-PD1+CTLA4, treatment, unlike the GL261 model and similar to GBM patients. We deeply characterized each model, including immune phenotyping and single-cell RNA-sequencing analyses. We also performed a cross-species comparison of human and mouse GBM and stromal cell phenotypes at the single-cell level. Our results show that new mouse models recapitulate the heterogeneity of glioma cell states observed in individual patients and, importantly, also the complex and immune suppressive TME. We demonstrate at the single-cell level that glioma-associated T and myeloid cell types are molecularly similar and conserved across species. Leveraging these data sets and experimental models, we are testing now novel immunotherapy targets for GBM.

EPCO-13. HUMAN ENDOGENOUS RETROVIRUS (HERV) PLAYS A CRITICAL ROLE IN ONCOGENIC MECHANISM INIDH1-WILD TYPE GLIOBLASTOMAS

Abstract IDH1-wildtype (WT) GBMs are the most common and aggressive tumors of the central nervous system (CNS), with a median survival rate of 12-15 months, and are resistant to clinical therapies due to their heterogenous molecular phenotype. In this study, we developed a pipeline to dissect the potential role of human endogenous retroviruses (HERVs) in patient derived glioma stem cells (GSCs) and integrated our findings with published epigenomic datasets for enhancers (H3K27ac), CTCF, and chromatin toplogy (Hi-C). We found high expression of several HERV transcripts in IDH1-WT GBMs as compared to IDH1-mutant variants. Epigenomic analysis of these upregulated HERVs showed association with both enhancer marks and CTCF deposition. Annotation of HERVs to their nearest genes, followed by Gene Set Enrichment Analysis (GSEA), identified involvement of actin-filament based mechanisms and regulation of immune phenotype. Integration of these findings identified MER61 (ERVMER61-1), a potential regulator of Interleukin-6 and showed alteration in chromatin looping and enhancer occupancy. To summarize, our work points to novel mechanisms of GBM pathogenesis, through involvement of HERVs in cell migratory and immune dysfunction, warranting further investigations into these oncogenic mechanism to identify and develop novel therapeutic options for GBM.

TMIC-73. SEX DIFFERENCES IN INTERFERON-GAMMA RESPONSES IN GLIOMA MICROENVIRONMENT

Abstract IFNγ is a cytokine with both pro- and anti-tumorigenic effects and its role in glioma immunotherapy resistance is not well understood. In the tumor microenvironment, T and NK cells produce IFNγ, which affects cellular phenotypes in most of the cancer and stromal cells since IFNγ receptor is ubiquitously expressed. IFNγ induces expression of molecules that confer resistance to immunotherapies, such as PDL1, PDL2, IDO1, and iNOS. The goal of this study is to elucidate cell type-specific responses to IFNγ in immune competent glioma models we developed that are resistant to immunotherapy. We intracranially injected male and female murine glioma cells into B6 and Ifng-/- honst mice to measure overall survival and perform immune phenotyping and single cell RNA-sequencing analyses. Ifnγ-/- hosts exhibited a shorter survival, and the proportion of GBM cells is higher in Ifng-/- hosts compared to wild type, indicating that the net effect of IFNγ signaling in glioma is anti-tumorigenic. In a recent study, we published that IFNγ-response pathway is enriched in the mesenchymal-like (MES-like) subtype of human GBM cells, compared to neutral-progenitor-like (NPC-like) and oligodendrocyte-progenitor-like (OPC-like) cells (Abdelfattah et al., 2022). To test our hypothesis that IFNγ signaling induces MES transition in glioma cells in vivo, we compared fractions of glioma cell subtypes in wildtype and Ifng-/- male and female hosts. In female Ifng-/- mice, MES-like GBM cell fraction was reduced while OPC-like fraction was increased, supporting our hypothesis. However, there was no difference in male hosts. Furthermore, gliomas grown in female B6 hosts showed higher infiltration of immune cells (CD45+), lymphocytes (CD3+) and fewer resident microglia cells (CD11b+CD45int), compared with Ifng-/- female hosts. In contrast, no significant difference in immune cell infiltration was observed between B6 vs Ifng-/- males. These results suggest an unanticipated and complex context-dependent role of IFNγ signaling in GBM, which include sex differences in its role in immune cell recruitment and MES transition in glioma cells.

A two-step, two-sample Mendelian randomization analysis investigating the interplay between gut microbiota, immune cells, and melanoma skin cancer.

This study aims to rigorously explore the potential causal relationships among gut microbiota (GM), immune cells, and melanoma skin cancer among participants from Europe, where this disease exhibits significant prevalence and profound societal impact. Using the genome-wide association analysis database, a double-sample Mendelian randomization (MR) analysis was drawn upon to investigate GM, immune cells, and melanoma skin cancer. The inverse variance weighted approach was applied to estimate the causal connections among these variables. A two-step MR analysis was employed to quantitatively gauge the impact of immune cells mediated GM on melanoma skin cancer. To address potential sources of bias, such as pleiotropy and heterogeneity, multiple analytical techniques were integrated. The MR analysis pinpointed 6 GM taxa related to either an augmented or declined risk of late-stage melanoma skin cancer. In the same vein, 32 immune cell phenotypes were noticed as correlates with modified risk of melanoma skin cancer. Our study also implies that the probable association between GM and melanoma could be facilitated by 5 immune cell phenotypes. The findings of our study underline certain GM taxa and immune cells as potential influencers on the onset and development of melanoma skin cancer. Importantly, our results spotlight 5 immune cell phenotypes as potential agents mediating this association.

Causal relationship between immune cells and hypopituitarism: bidirectional Mendelian randomization study

Hypopituitarism, one or more pituitary hormones inefficiently produced by the anterior pituitary or released from the posterior pituitary to adapt to the needs of the organism. Existing epidemiological data show that immune-mediated diffuse infiltration of the anterior pituitary is important in the development of hypopituitarism. However, the precise connection between immune cells and hypopituitarism remains unclear. This study aimed to elucidate the potential causal links between the 731 immune cell types and hypopituitarism risk. Based on data from a genome-wide association study (GWAS), a bidirectional two-sample Mendelian Randomization (MR) analysis was performed using five methods to explore the potential influence of immune cell phenotypes on hypopituitarism. Sensitivity analyses were conducted to examine the robustness of these findings. Our findings support that B cells, T cells, Tregs, dendritic cells, monocytes, and myeloid cells each have a bidirectional influence on hypopituitarism. One B-cell phenotype was associated with increased hypopituitarism risk, while two B-cell phenotypes play a protective role in hypopituitarism. Moreover, seven T-cell phenotypes demonstrate significant protective properties on hypopituitarism. Seven Tregs were associated with increased hypopituitarism risk. Furthermore, one monocyte was identified to be significantly associated with hypopituitarism risk. In addition, reverse MR analysis revealed that hypopituitarism was positively associated with 30 additional immune cell phenotypes and negatively associated with 17 immune cells. Our investigation shed light on the intricate potential relationship between immune cells and hypopituitarism via genetic methods, underscoring the immune-mediated pathway in hypopituitarism pathogenesis, thereby offering valuable insights for future clinical investigations.

M6A-related genes and their role in Parkinson's disease: Insights from machine learning and consensus clustering.

Parkinson disease (PD) is a chronic neurological disorder primarily characterized by a deficiency of dopamine in the brain. In recent years, numerous studies have highlighted the substantial influence of RNA N6-methyladenosine (m6A) regulators on various biological processes. Nevertheless, the specific contribution of m6A-related genes to the development and progression of PD remains uncertain. In this study, we performed a differential analysis of the GSE8397 dataset in the Gene Expression Omnibus database and selected important m6A-related genes. Candidate m6A-related genes were then screened using a random forest model to predict the risk of PD. A nomogram model was built based on the candidate m6A-related genes. By employing a consensus clustering method, PD was divided into different m6A clusters based on the selected significant m6A-related genes. Finally, we performed immune cell infiltration analysis to explore the immune infiltration between different clusters. We performed a differential analysis of the GSE8397 dataset in the Gene Expression Omnibus database and selected 11 important m6A-related genes. Four candidate m6A-related genes (YTH Domain Containing 2, heterogeneous nuclear ribonucleoprotein C, leucine-rich pentatricopeptide repeat motif containing protein and insulin-like growth factor binding protein-3) were then screened using a random forest model to predict the risk of PD. A nomogram model was built based on the 4 candidate m6A-related genes. The decision curve analysis indicated that patients can benefit from the nomogram model. By employing a consensus clustering method, PD was divided into 2 m6A clusters (cluster A and cluster B) based on the selected significant m6A-related genes. The immune cell infiltration analysis revealed that cluster A and cluster B exhibit distinct immune phenotypes. In conclusion, m6A-related genes play a significant role in the development of PD and our study on m6A clustering may potentially guide personalized treatment strategies for PD in the future.