bluuebunny/crossref_metadata_2025_split huggingface dataset:
Signaling flow cytometry: One-step staining for phenotyping and functional characterization of immune cells
Authors: Alonzo Emily , Manning Christopher , MacDonald Robert , Cunningham Lori 
Published: November 2024

Abstract
Understanding how signaling pathways in immune cells are modulated by various perturbations can reveal potential therapeutic targets and uncover mechanisms of therapeutic response. Immune cells are typically observed in the context of a heterogeneous mixture of cells, and techniques that enable single cell analysis, such as flow cytometry, are essential. However, observing changes in pathway activation by flow cytometry can be challenging as antibodies used for immune phenotyping are generally validated in live cell assays, while antibodies targeting signaling readouts usually require fixation and permeabilization. To simplify multiplexing with signaling and phenotyping readouts, we tested many commonly used immune phenotyping antibodies in multiple fixation and permeabilization protocols. We compiled the data into a table to enable design of flow cytometry panels that include both phenotyping and signaling antibodies and can be used as a single staining antibody mix on cells following fixation and permeabilization. To illustrate this functionality, we designed and generated data for two different panels on fixed, permeabilized cells. Using one panel, we identify B cells among PBMCs and observe phospho-Syk and phospho-CD79a following BCR activation. With the other panel, we compare phospho-STING across different myeloid subsets from PBMCs treated with the STING ligand cGAMP and observe upregulation of STING activation in classical monocytes, with very little change among DCs.
Nociceptor sensory neurons promote pneumonic sepsis during carbapenem-resistant Klebsiella pneumoniae lung infection.
Authors: Joshi Prabhu , Adhikari Sandeep , Judd Abigail , Baral Pankaj 
Published: November 2024

Abstract
Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a leading cause of Gram-negative pneumonia and pneumonic sepsis for which limited therapeutic options are available. The lungs are densely innervated by nociceptive sensory neurons (nociceptors), which mediate breathing, cough, and bronchoconstriction. However, the role of these neurons in host defense against CRKP-induced pneumonic sepsis is unknown. We found that nociceptors play a host deleterious role to promote pneumonic sepsis. Nociceptor-ablated mice showed an enhanced survival and bacterial clearance after CRKP lung infection. Furthermore, nociceptors ablation resulted in the increased influx of neutrophils and Ly6Chi monocytes and cytokine induction. Depletion of Ly6Chi monocytes, but not of neutrophils, impaired lung bacterial clearance and bacterial dissemination in nociceptor-ablated mice, indicating that Ly6Chi monocytes are a critical cellular population downstream of nociceptors to regulate pneumonic sepsis. Moreover, nociceptive neuropeptide calcitonin gene-related peptide (CGRP) suppressed induction of reactive oxygen species and intracellular bacterial killing. Collectively, our data suggest that the nociceptor neuronal signaling could be a therapeutic target for treating multidrug-resistant CRKP lung infections and pneumonic sepsis.
Supported by grants from NIH/NIGMS P20GM113117, American Lung Association, and JCRC Heart Research Award.
Investigating the role of Natural Killer cells in the control of latent Epstein-Barr virus infection
Authors: Pena Josselyn , Zhang Wenming , Esquivel Carlos , Martinez Olivia , Krams Sheri 
Published: November 2024

Abstract
Epstein–Barr virus (EBV) is a ubiquitous gamma-herpesvirus that persists as a chronic, asymptomatic infection in over 90% of the adult population with the virus transitioning to latency in a subset of memory B cells. Failure to control latent EBV can result in a variety of malignancies, including lymphoproliferative diseases. Studies in experimental models and humans suggest that NK cells are critical in the host defense against EBV. Previously, we demonstrated NK cells expressing the inhibitory receptor NKG2A were specifically able to recognize and kill autologous EBV-lymphoblastoid cell lines (EBV-LCL). We also showed that HLA-E-presented peptides derived from latent EBV proteins can impair NKG2A recognition and signaling, potentially leading to NK cell activation. To better understand the phenotype of NK cells that respond to latent EBV, we generated a panel of EBV-LCL and performed co-culture experiments with primary NK cells and autologous EBV-LCL. We developed a novel mass cytometry panel and found that in addition to NKG2A, EBV-LCL-responsive NK cells express increased NKp30 and CD32 and decreased CD16 which may contribute to NK cell activation and killing of EBV-LCL. Further, we generated HLA-E knockout EBV-LCL using CRISPR and determined that autologous NK cell cytotoxicity increased, suggesting that disrupting the NKG2A:HLA-E immune checkpoint axis enhances killing of EBV-infected B cells and would be an attractive therapeutic option for EBV-associated malignancies.
NFIL3/E4BP4 regulates the developmental stage-specific acquisition of basophil function
Authors: Park Jiyeon , Cho Yuri , Yang Dongchan , Yang Hanseul , Lee Daeyoup , Kubo Masato , Kang Suk-Jo 
Published: November 2024

Abstract
Basophils are essential effector cells in many allergic disorders, but the terminal developmental processes through which basophils acquire their unique functional properties are not fully understood. Here, we report the identification of a novel population of late-stage mitotic basophil precursor cells expressing high levels of the IgE receptor FcεRI alpha chain, termed ‘transitional basophils’ (tBasos). These tBasos produce type 2 cytokines more robustly than mature basophils, despite being less responsive to IgE stimulation. Transcriptome analysis revealed dynamic gene expression shifts during basophil development that enables precursor stages to proliferate and late-stage basophils to perform effector functions. Furthermore, we found that the transcription factor NFIL3/E4BP4 is essential for basophils to acquire IgE receptor-mediated signaling, cytokine secretion, and degranulation, critical basophil functions, as well as for the regulation of basophil-mediated allergic skin reactions. Collectively, our results unveil a new population of mitotic basophil precursors and elucidate the role of NFIL3/E4BP4 in establishing basophils’ IgE-mediated functions.
Inhibition of neutrophil elastase by the bacterial serine protease inhibitor ecotin in cystic fibrosis airway samples
Authors: Son Yeongseo , Fantone Kayla , Nothaft Harold , Stecenko Arlene , Szymanski Christine , Rada Balazs 
Published: November 2024

Abstract
Cystic fibrosis (CF), a prevalent fatal genetic condition in North America, disrupts respiratory function by impeding chloride ion transport across mucosal surfaces. This leads to the thickening of mucus, resulting in constant lung inflammation and elevated levels of neutrophils. Neutrophil elastase (NE), a potent serine protease crucial for host defense, is released by these neutrophils. However, in excess, NE can cause detrimental host tissue damage. Abundant in CF airways, NE is a key predictor of lung disease progression. While past studies have explored targeting NE in CF, no optimal inhibitors have reached clinics. Our study tested ecotin, a bacterial periplasmic serine protease inhibitor, for its potential to impede NE activity in CF airway samples and curb NE release from human neutrophils. Results show that ecotin significantly reduces NE activity in sputum samples obtained from several CF patients. Additionally, we found that ecotin inhibits NE release from neutrophils exposed to CF lung stimuli like Staphylococcus aureus bacteria. These results establish ecotin as a promising NE inhibitor in CF with clinical potential.
Adjuvant conditioning reprograms the innate and adaptive immune system through the NLRP3/IL-1 pathway
Authors: Boccia da Costa Thais , Pan Weikang , Fattori Victor , Rogers Michal , Zanoni Ivan , Cuenca Alex 
Published: November 2024

Abstract
While adjuvants are typically employed to boost immune responses, we have shown that multiple administrations of the adjuvant alum, adjuvant conditioning (AC), creates an immunosuppressive milieu that delays allograft rejection by expanding myeloid-derived suppressor cells (MDSCs). Since alum activates NLRP3, we performed AC in C57BL/6 and NLRP3-/- mice, with or without Anakinra before analyzing MDSC populations and immunizing mice with OVA. AC induces the expansion of highly suppressive MDSCs in the spleen of C57BL/6 mice, which is significantly decreased in the absence of NLRP3 (fold increase 2.1 vs 0.72, p&lt;0.0001), or when IL-1 signaling is blocked (fold increase 4.46 vs 0.48, p&lt;0.0001). AC before OVA immunization significantly decreased the proliferation of OVA-specific CD4 T cells and OVA-specific IgG titers compared to saline controls (p=0.0057, and p&lt;0.0001 respectively). In the absence of NLRP3, we observed the increased expansion of Th1, Th2, and Th17, and IFN-g and IL-4 production in OVA-specific CD4 T cells from AC-NLRP3-/- mice, and also higher OVA-IgG and IgE titers compared to the saline group. In a pancreatic alloislet transplantation model, AC-treated NLRP3-/- mice showed no difference in allograft survival compared to saline-treated controls, highlighting the requirement for NLRP3 signaling in AC. In summary, these data suggest that AC induces an immunosuppressive milieu through the NLRP3/IL-1 axis that shapes innate and adaptive immunity.
Respiratory syncytial virus infection alters the gut immune response to commensal bacteria
Authors: Siddikova Anisa , Brown Julia , Zeng Melody 
Published: November 2024

Abstract
Respiratory Syncytial Virus (RSV), characterized by inflammation of the bronchioles, is a major cause of hospitalizations among infants and older adults. Early-life gut dysbiosis, an imbalance of microbiota, has been linked with elevated risk of lung infections later in life. However, our understanding of the impact of respiratory infections on the gut and its long-term consequences is limited. We hypothesize that RSV infection in mice increases gut inflammation. To test this, we intranasally infected mice with RSV, collected blood and stool samples over the course of the infection, and analyzed immune cells in the lung and colon. We saw an increase in immune cells and cytokine production in both lung and colon of infected mice. We further found an increase in antibodies binding to commensal bacteria in the intestinal lumen, which was more prominent in the colon than in the small intestines, suggesting a tissue specific difference in response to RSV. Lastly, we saw gut microbiome changes in response to RSV, such as reduced Muribaculaceae and elevated Alistipes. Together, these data suggest that respiratory infections may disrupt homeostasis beyond the lung, and respiratory virus-induced changes to the gut could influence the risk of subsequent enteric infections or chronic conditions such as inflammatory bowel disease. A better understanding of how RSV influences the gut could help to identify patients at risk for these conditions as well as potential therapeutic targets.
InTraSeqTM: a multimodal single cell technology for simultaneous measurement of RNA and proteins in single cells to uncover new single cell biology
Authors: Ariss Majd 
Published: November 2024

Abstract
InTraSeqTM is a novel technology for single cell analysis that enables unbiased measurement of both RNA and proteins in individual cells. This innovative approach employs validated and functionally tested antibodies at CST, which allows the identification of cell subtypes and Signaling Pathways in single cells by measuring post-translational modifications (PTMs).
One of the significant advantages of using InTraSeqTM is its ability to quantify extracellular, cytoplasmic, and nuclear proteins, including transcription factors, and their PTMs while preserving the RNA and heterogeneity of the sample. This method offers an extensive view of the cellular mechanisms of complex biology, which was previously missing in single cell RNA sequencing datasets. Researchers can use InTraSeqTM to accurately characterize single cell acute and chronic responses, uncover new cell states, and more accurately analyze cell differentiation trajectories. Moreover, InTraSeqTM offers a streamlined protocol that requires only one hour of benchwork and allows for multi-day sample storage, making it a practical and efficient tool for high-throughput applications.
Revolutionary Selection, Activation, and Expansion with Microbubbles for CAR-T Cell Manufacturing
Authors: Badea Dominique , Huang Effie , Snow Tiffany , Chen Jesse , Roussey Jon , Ku Chia , Wegner Casey 
Published: November 2024

Abstract
Despite several chimeric antigen receptor-modified T cells (CAR-T) products being approved and commercialized, CAR-T cell therapy has manufacturing challenges. Conventional CAR-T therapies are manufactured by collecting apheresis material from a patient’s blood, then modifying the isolated T cells from the apheresis material to express a cancer cell-specific CAR, eventually infusing the CAR-T cells into a patient. Challenges include early exhaustion of the therapeutic cells, manual/laborious processes, and complex workflows. Akadeum’s innovative microbubble technology streamlines human T cell selection, activation, and expansion into a single step. Here, we demonstrate over 90% capture efficiency while reducing the selection process to under one hour. Because of the microbubbles’ natural buoyancy, T cell activation occurs at the top of the culture. As the T cells divide and the bound receptors are recycled, T cells fall to the bottom of the media, avoiding CD3/CD28 overstimulation. PD-1 expression is reduced throughout expansion compared to traditional activation workflows. T cell activation was not compromised, as evidenced by the upregulation of CD69, CD25, and HLA-DR+. Additionally, microbubble technology can increase the percentage of desired effector T cell populations. As CAR-T therapy expands, innovations like Akadeum's microbubble technology play a crucial role in overcoming manufacturing challenges and ensuring the development of high-quality, effective therapies.
A Fully Automated, Walkaway Flow Cytometry Workflow for Immune Cell Subsets Identification
Authors: Bowers Rita , Grazia Giulia , Maraman Jason , Lacaille Vashti , Weit Nicole 
Published: November 2024

Abstract
In the fast-paced competitive landscape of life science research, the adoption of an automated workflow offers the advantages of improved data reproducibility and increased throughput with less operator dependency, contributing to robust and efficient research studies.
We present here an automated flow cytometry workflow using a fully integrated system comprising a Biomek i7 Multichannel Workstation, a CytoFLEX LX flow cytometer, DURA Innovations dry reagents and automated data analysis using the Cytobank platform.
In our study, a DURAClone T cell subset dry reagent panel in a 96-well plate format was used to characterize maturation stages in T cells present in PBMC samples from 16 healthy donors. The walkaway workflow leveraged the Biomek i7 Multichannel liquid handler for automated sample preparation, automated sample acquisition on an integrated CytoFLEX LX flow cytometer, automated data upload to the Cytobank platform via the API (application programming interface) with automated data analysis performed using a pre-defined gating model applied to the newly created experiment, achieving auto-generated results ready for review.
Additional features and functionality within the Cytobank platform were leveraged to inspect results, apply dimensionality reduction algorithms, export tables of statistics and graph the final data.
By embracing automated workflows and advanced technologies, scientists can conduct their research more efficiently and reliably.
For Research Use Only
Pharmacological activation of LANCL receptors has three-fold benefit in metabolic dysfunction-associated steatohepatitis
Authors: Leber Andrew , Hontecillas Raquel , Tubau-Juni Nuria , Fitch Sarah , Bassaganya-Riera Josep 
Published: November 2024

Abstract
The LANCL family of receptors includes LANCL2, a membrane receptor associated with glycemic control and regulatory immune responses, and LANCL3, a mitochondrial receptor that is significantly downregulated in multiple inflammatory diseases. In metabolic dysfunction-associated steatohepatitis (MASH), LANCL3 expression is reduced more than two-fold in cholangiocytes, hepatic stellate cells and Kupffer cells relative to healthy control. Based on the mechanistic overlap between the LANCL receptors and MASH, we assessed loss of function and pharmacological activation in MASH relevant in vitro and in vivo systems. Loss of LANCL2 or LANCL3 resulted in increased production of IL-17 and TNF from CD4+ T cells. Production of COL1A1 in hepatic stellate cells increased three-fold after TGF-β stimulation in the KO relative to WT. In vivo, liver weight increased and glucose tolerance decreased in KO mice relative WT after high fat diet feeding for 12 weeks. In both carbon tetrachloride and choline deficient L-amino acid defined (CDAA) models of MASH, LANCL activation resulted in lower liver weight, triglycerides and fibrosis through measures of total, perivascular and bridging collagen. Treatment also resulted in a significant reduction of Th17 cells and TNF+ macrophages in the models of MASH. This data suggests that LANCL receptors are promising therapeutic mechanisms for MASH and other metabolic diseases through an ability to directly modulate the three hallmarks of MASH pathogenesis.
Immunaut: Immunophenotype Analysis with Machine Learning for LAIV Vaccine Response Prediction
Authors: Tomic Ivan 
Published: November 2024

Abstract
The Immunaut approach represents a groundbreaking application of machine learning in immunology, focusing on predicting immune responses to Live Attenuated Influenza Vaccines (LAIV). This novel method combines unsupervised machine learning techniques, including t-distributed Stochastic Neighbor Embedding (tSNE) and advanced clustering algorithms, with the Sequential Iterative Modeling OverNight (SIMON) platform for predictive analysis. By identifying and categorizing immunophenotypic groups based on baseline features, Immunaut offers a sophisticated, data-driven means of enhancing our understanding and prediction of vaccine efficacy. This approach promises to pave the way for more personalized vaccine strategies and a deeper insight into immune response mechanisms.
An IL-12 mRNA-LNP adjuvant durably enhances the CD8+ T cell response to mRNA vaccination
Authors: Aunins Emily , Phan Anthony , Cruz-Morales Elisa , Alameh Mohamad-Gabriel , Christian David , Hunter Christopher , Weissman Drew 
Published: November 2024

Abstract
The design of vaccines that can reliably induce protective CD8+ T cell activity has been considered a challenge for the vaccine field, but the use of viral vectors or mRNA encapsulated into lipid nanoparticles (LNPs) has overcome some of these issues. Because the cytokine interleukin-12 (IL-12) supports CD8+ T cell expansion and acquisition of effector functions, studies were performed to assess its contribution to the ability of mRNA vaccines to promote CD8+ T cell responses. mRNA-LNPs do not stimulate macrophage production of IL-12 in vitro nor does in vivo vaccination, and endogenous IL-12 is not required for the CD8+ T cell response to mRNA vaccination. However, the addition of IL-12 mRNA-LNPs into vaccination with LNPs that contain mRNA for the model antigen ovalbumin (OVA) results in enhanced OVA-specific CD8+ T cell expansion, improved acquisition of effector function, diversification of the CD8+ T cell effector pool through generation of a KLRG1hi effector subset and expanded effector and central memory CD8+ T cell populations. In both preventative vaccination against Listeria monocytogenes-OVA and therapeutic vaccination against B16 F0-OVA melanoma, addition of IL-12 mRNA-LNPs enhanced protection. Thus, modification of current mRNA vaccine formulations to induce IL-12 production provides a strategy to modify the CD8+ T cell response, enhance CD8+ T cell mediated protection and illustrates the utility of cytokine mRNA to tailor vaccine-induced immunity.
The ectromelia virulence factor C15 is a potent inhibitor of MHC-mediated T cell activation.
Authors: Carro Stephen , Hedgepeth Emma , Heard Mary , Peauroi Elise , Eisenlohr Laurence 
Published: November 2024

Abstract
Orthopoxviruses (OPXV), home to mammalian pathogens such as variola and Mpox, encode a myriad of immunomodulatory proteins, many of which are understudied, to promote pathogenesis. One protein family, the B22 family, are of interest as potential antiviral targets due to their conservation across pathogenic OPXV. B22 proteins are surface glycoproteins that inhibit T cell activation. In ectromelia virus, we previously found that its B22 protein, C15, is essential for lethality in BALB/c mice and inhibits CD4+ T cell activation both in vitro and ex vivo. However, the mechanism of C15 immune modulation remains unknown. To bridge this gap, here we define the breadth of C15-mediated T cell inhibition and explore its molecular mechanisms. Using T cell hybridomas, we found that C15 inhibits MHCIa/b- and MHCII-mediated T cell activation. This inhibition is not mediated by a secreted factor but is in effect upon expression by MHC-negative cells. This finding, along with the observation that C15 does not impact the surface density of peptide-MHC complexes, suggests that C15 acts directly on the T cell rather than the antigen-presenting cell. In vivo, we demonstrate that C15 significantly dampens the generation of antigen-specific cytotoxic effector CD8+ T cells (Teff), resulting in a significant decrease in KLRG1+CX3CR1+ Teff. Overall, our results offer novel insights into the molecular mechanisms of C15-mediated T cell inhibition and the modulation of adaptive immunity by OPXV.
Antigen-Specific Germinal Center Tfh cells are highly heterogenous in lymph nodes and Tfh1/Tfh17 cells are associated with viral load levels during chronic SHIV infection
Authors: Govindaraj Sakthivel , Styles Tiffany , Ibegbu Chris , Amara Rama , Velu Vijayakumar 
Published: November 2024

Abstract
HIV infection leads to alterations in HIV-specific CD4 T cells including increased expression of inhibitory receptors and skewing toward Tfh cell signature. Tfh cells have been shown to increase during chronic HIV infection. However, the heterogeneous nature of antigen-specific GC-Tfh cells and the relative contribution of specific GC-Tfh subset to viral persistence is not understood in HIV/SIV infection.
Here, we characterized the phenotype and function of antigen-specific GC-Tfh cells based on the expression of chemokine receptors, Th1 (CXCR3), Th2 (CCR4), and Th17 (CCR6) along with activation-induced markers (AIM assay) CD25, OX40 and 41BB in the lymph nodes following chronic SHIV1157ipd3N4 infection in rhesus macaques.
In SHIV naïve RM, only a small fraction of total GC-Tfh expressed CXCR3, CCR4, and CCR6. During chronic SHIV infection, there was an increase in GC-Tfh cells. Interestingly the majority of antigen-specific GC-Tfh cells expressed CXCR3 (Tfh1), while a significant proportion of them also expressed CCR4+ (Tfh2) and CCR6+ (Tfh17) cells. These cells expressed OX40, 41BB, and CD25 at higher levels than non-Tfh cells. Importantly, the expansion of GC-Tfh1 and GC-Tfh-17 cells is associated with high viremia.
These data demonstrate that chronic SHIV infection promotes the expansion of antigen-specific GC-Tfh cells with heterogeneous populations, which are distinct from conventional GC-Tfh, and associated with higher viral RNA levels during chronic SHIV infection.