bluuebunny/crossref_metadata_2025_split huggingface dataset:
Elucidating the Transcriptional Role of the Nuclear Receptor RORα in CD8+ T Cell Memory vs Effector Cell Development
Authors: Chuck Jonathan , Puvvala Avanti , Campbell Sean , Solt Laura 
Published: November 2024

Abstract
CD8+ T cells play a critical role in controlling viral infections and cancer and have the potential to be harnessed for the development of new therapeutic strategies. Defining the transcriptional regulatory networks that control CD8+ T cell differentiation and function is of great importance to achieve these goals. The retinoic acid receptor-related orphan receptor alpha (RORα), a member of the nuclear receptor superfamily of ligand-regulated transcription factors, has emerged as a potential transcriptional regulator of CD8+ T cell immunity. However, exactly how RORα functions in these processes is poorly understood. To this end we seek to identify the transcriptional role of RORα in regulating the differentiation of memory CD8+ T cells. Utilizing a mouse model with T cell-specific deletion of RORα we identify that RORα deficiency in CD8+ T cells leads to increased IFNγ production and increased cell-specific cytolytic function in vitro. Conversely, RORα overexpression results in decreased IFNγ production and defects in proliferative capacity in vitro. Our in vivo data further reveals that deletion of RORα in CD8+ T cells may affect the trafficking of these cells to non-lymphoid tissue and play a role in the development of tissue resident memory (TRM) cells. Our study provides important insights into the role of RORα in regulating CD8+ T cell function and highlights the potential for targeting nuclear receptors in the development of novel cell-based therapeutics.
Cellular immune responses in the guinea pig model with a 9-color phenotypic flow cytometry panel
Authors: Anthony Scott , Marketon Anthony , Pahar Bapi , Rivera Deja , Barr Bobbi , Bernbaum-Cutler Rebecca , Caravan Peter , de la Torre Juan Carlos , Mani Venkatesh , Carey Brian 
Published: November 2024

Abstract
Guinea pigs serve as a valuable animal model for many aspects of infectious disease research. A primary limitation of the guinea pig model is the lack of a robust toolbox to probe the underlying immune responses in these animals. We developed an optimized 9-color flow cytometric assay for immunophenotyping peripheral and tissue-associated immune cells in the guinea pig. Using this assay, we can delineate CD45+ cells as well as monocyte and macrophage populations, in addition to further differentiating lymphocyte populations of B and T cells. Importantly, we found that this panel is able to detect these populations in both inbred Strain 13 and outbred Hartley guinea pigs in whole blood and from fresh and frozen cells isolated from several peripheral tissues. We utilized this panel to separately interrogate the immune responses in whole blood and spleen tissue samples after exposure of Hartley guinea pigs with Ebola virus (EBOV) and Strain 13 or Hartley guinea pigs with Lassa virus (LASV). We believe this flow cytometry panel will serve as an important tool to interrogate the underlying immune responses to risk group 4 pathogens in addition to numerous other infectious diseases with the guinea pig model.
A Simplified Workflow for Flow Cytometric Analysis of Extracellular Vesicles on the Invitrogen™ Attune™ NxT Flow Cytometer
Authors: Ellis Samantha , Zahner Sonja , Nguyen Chau , Defelippi Kendall , Li Xiao 
Published: November 2024

Abstract
Extracellular vesicles (EV) are miniscule particles composed of a bilipid membrane that are released by all cells. In recent years, it has become clear that EV play an important role in cell-cell communication by shuttling their cargo of proteins, lipids, and nucleic acids. As their content can be indicative of the health of their cell of origin and ability to deliver functional biomolecules, EV have become an increasingly popular topic of research for their diagnostic and therapeutic potential.
EV vary in size, surface markers, and content. Their heterogenous nature introduces challenges for analysis when using technologies that were developed for cells. In flow cytometry, EV have a lower refractive index and paucity of epitopes comparative to cells, which hampers detection. In addition, few commercially available antibodies have been specifically validated for EV flow. This combined with the need for tedious isolation processes and a limited range of instruments capable of small particle detection can make EV flow cytometry a daunting endeavor.
Despite this, flow cytometry holds promise as an accessible approach to analyze EV. Here, we present a streamlined protocol of EV characterization by flow cytometry in which we adapted the Invitrogen™ CELLection™ Pan Mouse IgG Kit for EV capture and staining and optimized the Invitrogen™ Attune™ NxT Flow Cytometer for small particle resolution to validate monoclonal antibodies targeting EV from human plasma. For research use only.
Development of a High-Throughput Image Cytometric Screening Method as a Research Tool for Immunophenotypic Characterization of Patient Samples from Clinical Studies
Authors: Chan Leo , Nitta Carolina , Patel Samir , McDonald James , Mohammed Hamza , Hem Sopaul , Pierce Mackenzie , Parthasarathy Vaish , Hernandez Veronica , Stuckey Tyanna , Lin Bo , Reading Julian 
Published: November 2024

Abstract
Immune cell characterization from patients undergoing therapeutic treatments in clinical research is critical for understanding disease progression and treatment efficacy. Recently, a novel image cytometry system (Cellaca™ PLX Image Cytometer, Revvity, Inc.) was developed to complement flow cytometry immunophenotyping. Initial studies investigated the feasibility of identifying T-cell, B-cell, NK-cell, and monocyte populations in healthy donors in comparison to flow cytometry. Donor PBMCs were stained with three panels of surface markers: B-cell with CD3-KIRAVIA Blue 520 ™(KB) and CD19-PE; NK-cell with CD3-KB and CD56-PE; and monocyte with CD3-KB and CD14-PE. Stained samples were acquired on the image cytometer and the CytoFLEX (Beckman Coulter, Inc.). Results show similar population percentages within 5%. Furthermore, we tested the screening method using patients enrolled in autoimmunity (rheumatoid arthritis) and oncology (multiple myeloma) disease cohorts. 48 primary PBMC samples were analyzed with Panel 1 of CD3-KB, CD56-PE, and CD14-APC, and Panel 2 of CD3-KB, CD56-PE, and CD19-APC. We validated the image cytometry method by comparing the Cellaca PLX to the Aurora (Cytek™). Results demonstrated comparable CD3, CD14, CD19, and CD56 cell populations between both instruments, showing ~ 5 – 10% differences. The proposed image cytometry method provides a novel high-throughput research tool to streamline the immunophenotyping workflow for patient sample characterization.
ThresholdR: An Automated Method for Thresholding CITE-seq-based Antibody Expression Data for Immune Cells
Authors: Oliaeimotlagh Mohammad , Kumar Sunil , Khan Amir , Silas Armstrong Suthahar Sujit , Suryawanshi Vasantika , Makings Jeffrey , Durant Christopher , Drago Fabrizio , Taylor Angela , McNamara Coleen , Lanier Lewis , Hedrick Catherine , Ley Klaus 
Published: November 2024

Abstract
Cellular Indexing of Transcriptomes and Epitopes (CITE-Seq) enriches single cell transcriptomic insights by incorporating information about the cell surface phenotype through the application of oligonucleotide-tagged monoclonal antibodies. Similar to observations in flow cytometry, surface protein quantification in CITE-Seq introduces technical noise, potentially obscuring underlying biological processes and giving rise to misleading results. This noise originates from ambient antibodies within the reaction compartment (such as droplets or wells) as well as non-specific binding. Here, we present ThresholdR, an R-based automated tool, to reliably and systematically find the threshold for each antibody under the experimental conditions used in each experiment. We show that ThresholdR helps identification of cell types and enhances the presentation of biological differences across cell populations. Flow cytometry validation confirmed the proportions of cell populations per patient identified by ThresholdR, demonstrating strong and statistically significant correlations. Furthermore, our findings reveal that cell type-specific noise stems from the non-specific binding of antibodies, with larger cells, such as monocytes, exhibiting higher levels of noise compared to other cell types. We systematically assess the performance of ThresholdR across diverse datasets and platforms, underscoring the agreement between individual cell data and well-established surface markers.
Highly efficient non-viral delivery of macromolecules to unactivated human T cells using microfluidic transfection
Authors: Chehal Manreet , Ouellet Eric , Binson Elinor , Nguyen Quan , Liao Tina , Chau Phillip , Chen Kris , Lambertz Ulrike , Yu Jessie , Chen Kevin , Loughhead Scott , Dalton Bob , Eaves Allen , Louis Sharon , Kokaji Andy 
Published: November 2024

Abstract
Traditional methods for transfecting unactivated human T cells are limited in their delivery efficiency while maintaining cell viability, phenotype, and function. To address these challenges, we developed the CellPore™ Transfection System. This microfluidic platform creates transient disruptions in the plasma membrane via pressure-induced mechanical deformation, allowing for cytosolic delivery.
First, we developed a workflow to optimize delivery pressure to T cells using FITC-dextran. This optimized pressure was used to deliver mRNA for expression of eGFP (91.8%) and mCherry (92.9%), and Cas9 RNP complexes targeting B2M and TRAC, with knockout efficiencies of 74.0% MHC-I and 93.9% TCRαβ, respectively. Importantly, while electroporation of T cells resulted in increased expression of the activation marker CD69 (41.2%) and proinflammatory cytokines (77.7 pg/mL IL-2, 8.8 pg/mL IFNγ), CellPore™-transfected T cells maintained an unactivated phenotype (0.8% CD69+, 0.1 pg/ml IL-2 and 0.2 pg/ml IFNγ) and were capable of downstream activation/expansion via CD3, CD28, and CD2 cross-linking. Using this optimized workflow, efficient cytosolic delivery of FITC-dextran to human CD4 (97.4%), CD8 (96.6%) and regulatory (98.3%) T cells was also achieved.
The CellPore™ Transfection System enables robust and efficient delivery of cargo to T cells without impacting cell function in a simple workflow that can easily be integrated into research protocols.
Fast and Convenient Human Immune Profiling of Fresh and Long-Term Stabilized Whole Blood Samples Enabled With CyTOF
Authors: Watson Jessica , Hasan Shakir , Li Stephen , Loh Christina 
Published: November 2024

Abstract
Whole blood (WB) analysis within 24 hours of collection is crucial for monitoring clinical efficacy of therapies. WB collection and cytometric analysis are often performed at different sites, leading to significant delays. Fixation of WB with stabilizer reagents can overcome these challenges. However, not all antibody panels are compatible with these reagents. A high-parameter CyTOF™ panel was created to be compatible with commercial WB stabilizers to enable fast and convenient human immune profiling.
The CyTOF panel contains 20 metal-tagged antibodies to identify over 30 immune cell populations in a single tube of fresh or stabilized WB. There are more than 30 open channels for additional markers. Flexibility of the panel and its workflow was shown using two methods of stabilization, in which WB is either stained followed by freeze storage or WB is first frozen then later stained. To reduce technical variability in staining and acquisition, the CyTOF panel was pooled and frozen as single-use aliquots ahead of time, and samples were barcoded and acquired as a single tube.
The CyTOF panel for broad immune profiling overcomes analysis challenges caused by logistical delays as WB samples can be frozen with various workflows. Furthermore, freezing antibody cocktails is a unique feature of CyTOF flow cytometry, ensuring staining consistency. Thus, CyTOF workflows enable fast and convenient cytometry of WB samples.
For Research Use Only. Not for use in diagnostic procedures.
Systemic impact of melanoma on bone marrow-derived immune cells
Authors: Carey Alexis , Palm Cheyenne , Chhabra Yash , Weeraratna Ashani 
Published: November 2024

Abstract
Melanoma accounts for the most skin cancer-related deaths in the United States. As patients age, antitumor responses decline, making age a negative independent prognostic factor of overall survival in melanoma. Delving into age-associated changes in lymphoid organs, such as increased bone marrow (BM) adiposity, could improve the understanding of the aged antitumor response. We hypothesize that the age-related increase in BM fat drives the functional decline of immune cells, ultimately hindering the systemic antitumor response to melanoma. We utilized flow cytometry to analyze melanoma and age-related alterations to the BM, blood, and tumor immune cell composition. Our preliminary data suggests that mice challenged with melanoma systemically increased granulocytes. However, only aged tumors had an increase in granulocyte infiltration. Meanwhile, T cells significantly decreased in the BM of tumor-bearing mice. Additionally, there was a significant increase in lipids in the BM T cells from non-tumor-bearing aged mice and a reduction in these lipids when mice were challenged with tumor. Thus, there appears to be an age-dependent systemic immune response when the mice are burdened with melanoma. As a future direction, we plan to investigate the role of the aged BM niche on the effectiveness of immunotherapies. These insights could lead to improved outcomes for elderly patients with melanoma.
Exploring the Biomedical Potential of MXenes in Macrophage Phenotype Identification and Tracking
Authors: Longo Giorgia , Giro Linda , Fusco Laura , Resto-Trujillo Natalya , Chacon Benjamin , Shuck Christopher , Pigazzi Martina , Gogotsi Yuri , Ley Klaus , Orecchioni Marco , Delogu Lucia 
Published: November 2024

Abstract
Macrophages are innate immune cells with varying phenotypes and functions that are influenced by their microenvironment. The distinction between the classical M1 and M2 phenotypes has been crucial in understanding their involvement in inflammation. Recently single-cell technologies have revealed a complex range of macrophage activation states, each contributing differently to maintaining physiological balance and affecting the development and progression of diseases. Identifying the origins and function of these macrophage subsets remains a complex challenge. MXenes, a category of two-dimensional transition metal carbides and nitrides, show promise in the biomedical field owing to their notable features, such as biocompatibility. MXenes' unique chemical properties allow for their detection at the single-cell and tissue level using mass cytometry (CyTOF), which shows great promise for biomedical applications. We found that MXenes can be internalized by myeloid cell precursors, monocytes, and macrophages without affecting their survival or differentiation. Early evaluations of MXenes uptake by immune cells have shown their detectability even after 30 minutes of incubation, attributable to their chemistry and impressive signal-to-noise ratio at CyTOF. These characteristics position MXenes as effective tools for precise single-cell labeling and in vivo monitoring of monocyte-derived macrophage phenotypes across various tissues and in response to specific stimuli or diseases.
Assessment of the Fetal Gut Immunome after Decidua-Placental Inflammation
Authors: Vazquez Jessica , Keding Logan , Schotzko Michele , Antony Kathleen , Racine Jenna , Simmons Heather , Shah Dinesh , Golos Thaddeus 
Published: November 2024

Abstract
Exposure to maternal inflammation in utero has been shown to impact the long-term immunological health of the fetus.
Tisseel (n = 5), a surgical glue, or saline (n = 3) was injected directly into the placenta of pregnant (~GD90) rhesus macaques. C-sections were performed at ~GD150. Fetal colon and jejunum were collected at necropsy and mononuclear cell (MCs) were isolated. Amniotic fluid was collected and cytokine levels assessed by cytometric bead analysis. Colon and jejunum MCs were labeled with fluorochrome-conjugated antibodies against markers for B cells, Innate Lymphoid Cells (ILCs), and T cells. Data was acquired using the Cytek Aurora spectral cytometer and analyzed on SpectroFlo (v3.0) and pre-gated to Live CD45+ cells in FlowJo (v10.8.1). Pre-gated FCS files were then clustered and visualized within R package Specter (v1.0.0). Post-analysis was done within Specter, with appropriate statistical test, p &lt; 0.05 considered significant.
Tisseel fetuses had a unique population of activated ILC3s in the colon. In the fetal jejunum, we found that the Tisseel fetuses, similarly had an activated Tbet+ T cell population. In addition, Tisseel fetuses also had a unique, activated ILC population. Interestingly, we did not find any differences in cytokine levels in the amniotic fluid of these pregnancies.
Overall, decidual/placental inflammation led to disturbance of the fetal gut immunome. The mechanism by which these changes are occurring are still under investigation.
Altered mitochondrial homeostasis in SLE neutrophils promotes aberrant NET release but blunts NETosis during infection.
Authors: Reber Callista , Wise Ashley , TenBarge Eden , Mendonca Jessica , McDaniel Sarah , Mennen Ellie , Holder Bailey , Belevsca Eva , Ferris Jennifer , Burcham Lindsey , Bieber Jeffry , Monteith Andrew 
Published: November 2024

Abstract
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a lack of immune tolerance to nuclear antigens leading to systemic inflammation. Patients with SLE are more susceptible to bacterial infections, which is thought to be the result of immunosuppressants used to treat their disease. However, we showed that SLE neutrophils are impaired in their response to bacterial infection. While the unique cell death process resulting in the formation of neutrophil extracellular traps (NET; NETosis) has been attributed to antagonizing SLE pathology, we demonstrated that SLE neutrophils fail to undergo NETosis in response to the bacterial pathogens. Herein, we identified that production of lactate by phylogenetically distinct bacterial pathogens caused increased generation of mitochondrial superoxide and triggered NET release. In contrast, SLE neutrophils displayed an altered metabolic state that rendered them incapable of sensing bacterial lactate. As such, SLE neutrophils failed to undergo NETosis in response to the bacterial pathogen Staphylococcus aureus and underwent aberrant NET release upon stimulation with apoptotic debris. These results indicate that NET formation in response to bacteria may be distinct from pathways promoting heightened NETosis in SLE. Furthermore, deciphering the metabolic disruptions driving inappropriate NET formation in SLE may lead to clinically actionable biomarkers that limit accumulation of nuclear antigens and restores host defense.
Metabolic modulation unleashes immune vigor in murine pancreas tumors
Authors: Poimenidou Maria , Le Janet , Darnell Chad , AbuEid Mahmoud , McAllister Donna , Dwinell Michael 
Published: November 2024

Abstract
Our study investigates the transformation of the pancreatic ductal adenocarcinoma tumor microenvironment from an immune-excluded to an immune-inflamed state using metabolic inhibitors metformin and mitochondria-selective conjugates, MMe and pCF3-MMe. In murine models of pancreas cancer, MMe and pCF3-MMe exhibited potent inhibition of tumor growth. Flow cytometry indicated increased levels of total T cells, activated CD8+, and NK T cells in MMe and pCF3-MMe treated mice. The anti-tumor effects of these metabolic inhibitors were reversed upon CD8+ T cell immunodepletion. In vitro experiments demonstrated elevated levels of pro-inflammatory chemokines in MMe-treated tumor cells, aligned with increased CD8+ T cell infiltration. Furthermore, metabolic inhibitor treatment activated both helper CD4+ and cytolytic CD8+ T cells, with the latter demonstrating increased glycolysis and acidification rates indicative of enhanced activation. Flow cytometry corroborated these findings, revealing heightened proliferation and activation markers on treated T cells. In addition to the direct effects on T cells, immunofluorescence microscopy suggested the activation of the mitochondrial antiviral-signaling protein (MAVS) pathway in tumor cells and an increase in apoptosis as indicated by the significant increase in caspase 3/7. Our findings highlight the potential of metabolic inhibitors to activate anti-tumor immune responses directly and indirectly in murine pancreas tumors.
Single cell RNA sequencing analysis of tumor infiltrating γδ T cells in murine and human glioblastoma.
Authors: LA JEONGWOO , Kang In , Lee Heung Kyu 
Published: November 2024

Abstract
Glioblastoma multiforme (GBM) is the most commonly occurring malignant brain tumor. The anatomical location and the immunosuppressive TME have brought disappointing results for multiple therapeutics approaches, including immune checkpoint inhibitors (ICIs). γδ T cells are an attractive target for adoptive cell therapies (ACTs) due to their MHCI-independent tumor killing mechanism and GVHD-Free nature. Thus, investigating GBM infiltrating γδ T cells can provide insight into the pro-tumoral and anti-tumoral roles of γδ T cells and provide potential targets for gene engineering prior to ACT. Our Single-cell RNA sequencing (scRNA-seq) and flow cytometric data demonstrate that GBM harbors subtypes of γδ T cells of different origins and functions. γδ T cells from the meninges produce IL-17A and are enriched for genes related to Rorγt programme and immune checkpoint molecules. On the contrary, blood-borne γδ T cells produce IFN-γ and express genes related to cytotoxicity, co-stimulation and activating NK receptors. Functional dichotomy of γδ T cell is also observed in human GBM, with Areg producing tumor-promoting subset and Gzmb producing cytotoxic subset. Interaction analysis reveals that these subtypes of γδ T cell strongly interact with myeloid cells through unique receptor-ligand interaction. These finding reveal the comprehensive role of γδ T cells during cancer progression and provide insights into potential gene-editing targets for effective ACTs.
Type I IFN regulates neutrophils responses to murine beta-coronavirus infection
Authors: Worley Matthew , Gong Heng , Carver Kyle , Deng Jane 
Published: November 2024

Abstract
Introduction: Type I IFNs play a critical role in the immune response to viral infections. Suppressed type I IFN levels are predictive of severe SARS-CoV-2 infections. Given the critical role of neutrophils in viral-mediated lung injury, we investigated how type I IFNs regulated neutrophil responses during pulmonary coronaviral infections.
Methods: IFNAR-/- and C57BL/6 (WT) mice were infected with 2,500PFU of murine beta-coronavirus (MHV-1) and survival determined. At day 3 post infection, cellular recruitment, cytokine profiles and markers of lung injury were evaluated. Neutrophils were evaluated for activation markers, ROS generation and NET formation.
Results: All IFNAR-/- mice succumbed within 5 days versus 100% survival for WT mice. IFNAR-/- mice had higher pulmonary levels of GM-CSF (p=0.0079) and 3-fold more recruited neutrophils (p=0.0159) and monocytes (p=0.016). IFNAR-/- lung neutrophils had significantly lower TLR3, TLR4, TLR7, TLR9, CD88, CXCR2, CD69 (p&lt;0.05 for all) but increased levels of PDL-1. IFNAR-/- neutrophils isolated during infection showed significantly decreased ROS and NETs compared to WT.
Conclusion:IFNAR-/- mice are highly susceptible to MHV-1, displaying elevated neutrophil recruitment with lower frequency of activation markers, reduced ROS and NETs, and increased expression of PDL-1. Our findings demonstrate mechanisms by which inadequate type I IFN levels subvert critical aspects of neutrophil responses during severe viral respiratory infections.