bluuebunny/crossref_metadata_2025_split huggingface dataset:
Single cell transcriptomics of in vivo differentiating antibody-secreting cells reveals a divergent activated B cell program dependent on IRF4
Authors: Scharer Christopher , Patterson Dillon , Mi Tian , Price Madeline , Hicks Sakeenah L. , Boss Jeremy M 
Published: January 2023

Abstract
The generation of antibody-secreting cells (ASC) requires reprogramming the B cell transcriptome, epigenome, and metabolic potential to support the processes necessary to synthesize and secrete antibodies. These molecular changes have largely been studied at defined stages representing naive B cells, activated B cells, and ASC. To further refine when ASC fate decisions occur, we adoptively transferred CTV labeled B cells and monitored differentiation to both type I and II T cell independent antigens. At the peak of ASC responses, transferred cells were phenotyped by flow cytometry and isolated for single-cell RNA-seq. These data captured a continuum of responding B cells representing all stages of differentiation, with the largest heterogeneity observed among actB. Computationally ordering cells along differentiation trajectories, revealed divergent actB differentiation paths, with one branch leading to ASC formation that was dependent on IRF4. Moreover, cells that followed this trajectory downregulated the surface marker L-selectin and could be separated from cells that followed the alternative non-ASC differentiation branch. Indeed, isolation of actB by L-selectin status confirmed that L-selectin negative cells were ten times more likely to form ASC in culture. In summary these data provide insights into the cell division kinetics, molecular heterogeneity, and differentiation paths that lead to ASC formation.
Intrinsic Impairment of CD8 T Cells Induced by Helminth Coinfection
Authors: Rovira Eliezer , El-Naccache Darine W. , Zhao Yanlin , Gause William , Yap George S 
Published: January 2023

Abstract
Although it is known that helminth-triggered type 2 response increases host vulnerability to coinfections and potentially decreases vaccine efficacy, the exact mechanism(s) for the helminth-induced immunomodulation is still a fundamental question in the field. We have previously reported that CD44hiCD62L−KLRG1+ effector CD8 T cells from mice coinfected with Heligmosomoides polygyrus (H. polygyrus) and Toxoplasma gondii (T. gondii) exhibit a suppressed effector differentiation and impaired IFNγ production upon in vitro re-stimulation with toxoplasma antigens. This impairment was accompanied by a decrease in both, mitochondrial biomass and gene expression of the mitochondrial transcription factor, TFB1M. Further analysis of CD44hiT. gondii-specific CD8 T cells via single cell RNAseq, revealed an effector CD8 T cell subset from coinfected mouse that exhibit down-regulation of Cox17 and Atp5k transcripts. Both of these transcripts encode for proteins that belong to the electron transport chain multimeric complexes III and V, respectively. Taken altogether, our data suggest a potential metabolic-dependent mechanism of effector CD8 T cell insufficiency induced by helminth-coinfection. We hypothesize that helminth infection results in an impairment of CD8 T cell metabolic reprogramming, thus, affecting their effector development and function in response to T. gondii infection. The aim of this work is to increase the understanding of the helminth-associated immunomodulatory mechanism of CD8 T cells with the objective of finding therapeutic ways to restore CD8 T cells effector functions to improve vaccine efficacy in helminth-affected subpopulations.
Targeting CD49a+PBX1+ NK cells in recurrent spontaneous abortion immunotherapy
Authors: Yonggang Zhou , Binqing Fu , Haiming Wei 
Published: January 2023

Abstract

Objective
In the first trimester, CD49a+ natural killer (NK) cells are the main immune cells at the maternal-fetal interface. We have demonstrated that uterine NK cells have important physiological functions that can secrete various growth factors to promote fetal development. However, the functional subsets and transcriptional regulation mechanisms of NK cells expressing GPFs are unclear.

Result
Here, we have identified CD49a+PBX1+ NK cells as a unique subset in which the transcription factor, PBX1, upregulates the expression of the GPFs, pleiotrophin and osteoglycin, to promote fetal development at the maternal-fetal interface of both humans and mice. The results of ChIP experiments with anti-PBX1 antibody in decidual NK cells shown that PBX1 regulates the transcription of pleiotrophin and osteoglycin genes via direct binding. Inactivation of Pbx1 in mouse NK cells caused impaired fetal development due to a significantly decrease in the expression of GPFs. The results of exome sequencing of decidual NK cells from patients with unexplained recurrent spontaneous abortion (URSA), revealed decreased expression of PBX1 correlated with fetal growth restriction and pregnancy failure, suggesting that the impaired PBX1 gene in decidual NK cells is a potential biomarker for URSA.

Conclusion
Our study revealed that the transcriptional regulation mechanism in NK cells at the maternal-fetal interface, and suggested that impaired CD49a+PBX1+ NK cells contribute to the etiology of URSA, and provided the basis for improved approaches to NK cell-based immunotherapy for pregnancy related diseases.
Myeloid cells recruit colonic TCRab+ Intraepithelial Lymphocytes through Sphingosine-1-phosphate
Authors: Lefferts Adam R , Regner Emilie , Sansone-Poe Danielle , Norman Eric , Schultz Hannah , Kuhn Kristine 
Published: January 2023

Abstract
Intraepithelial Lymphocytes (IELs) are T-cells that exist in direct contact with the gut epithelium. IELs are important for barrier function; however, the specific pathway(s) by which they are recruited to the colonic epithelium remain unknown. To begin to address this question, we first performed a microarray comparing cell surface receptor expression in IELs versus lamina propria T cells. The array identified sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) as highly enriched in the IEL population. We also observed that tissue S1P levels are significantly lower in animals treated with antibiotics and in germ-free animals, both of which have reduced IEL numbers. Blockade of the S1P signaling axis during recolonization of an antibiotic-treated mice using FTY720 significantly reduced levels of TCRab+ IELs, but not TCRgd+ IELs. Next, since IEL recruitment has been shown to be Myd88 dependent, we used a series of lineage specific genetically deficient mice to identify the cellular source of the S1P. We found that LysM-Cre x Myd88fl/fl mice had significantly reduced TCRab+ IEL numbers in the colon. Additionally, Cre-negative but not LysM-Cre+ Myd88fl/fl deficient bone-marrow derived macrophages upregulate the primary kinase that generates S1P, sphk1, in response to stimulation with cecal contents, and induce T cell migration in a trans-well system in an S1P dependent manner. These findings indicate that TCRab+ IELs are recruited to the colonic epithelium by myeloid lineage cells via S1P in response to Myd88 mediated detection of microbial products. This is a new mechanism by which the microbiome influences colonic immunity, and indicates that TCRab+ IELs are dynamically recruited to the epithelium to mediate barrier function.
Th17 cell production of amphregulin contributes to the intestinal homeostasis
Authors: Zhao Xiaojing , Yang Wenjing , Yu Yanbo , Yu Yu , Zhou Zheng , Bilotta Anthony J , Yao Suxia , Cong Yingzi 
Published: January 2023

Abstract
Th17 cells have been implicated in the pathogenesis of inflammatory bowel diseases. Despite their inflammatory nature, Th17 cells are highly enriched in the intestines under homeostatic conditions. However, it remains unclear as to their exact role during intestinal homeostasis. We found that CBir1 TCR transgenic (CBir1 Tg) mice, which are specific for an immunodominant microbiota antigen CBir1 flagellin, demonstrated a higher levels of Th17 cells and higher amounts of amphiregulin (Areg), which has been recently shown to be highly protective at the mucosal surfaces, in the intestines compared to wild-type (WT) mice. Administration of Areg to WT mice led to decreased intestinal injury and higher level of tight-junction protein expression upon DSS insult. We further showed that Th17 cells produced higher levels of Areg compared to Th1 cells, and Areg inhibited T cell proliferation. Consistently, TCRβ/δ−/− mice transferred with Th17 cells developed less severe colitis compared with mice received Th1 cells. Additionally, TGF-β and IL-6, but not IL-23, the critical cytokines for Th17 development, promoted Areg expression in T cells in a time and dose dependent manner. Treatment with IL-21, but not IL-17, which are Th17 signature cytokines, upregulated Areg. Consistently, IL-21R−/− T cells produced lower levels of Areg. Mechanically, Stat3 mediates Areg expression in T cells, in that IL-6 promoted Stat3 activation, and IL-6 did not induce Areg expression in Stat3−/− T cells. Furthermore, Areg expression was lower in Stat3−/− T cells compared with WT T cells under Th17 polarization conditions. Collectively, these findings reveal that intestinal Th17 cells contribute to intestinal homeostasis and immune regulation by production of Areg.
Temporal changes in cellular sources of CXC chemokines control neutrophil recruitment during Citrobacter rodentium infection
Authors: Cai Baiyi , Zindl Carlene L. , Silberger Daniel J. , Figge David A. , Singer Jeffery R. , Merz Simon F , Gunzer Matthias , Weaver Casey T. 
Published: January 2023

Abstract
Enteropathogenic E. coli (EPEC) is one of the leading causes of diarrheal disease among children. Citrobacter rodentium (C.r) is a mouse model for EPEC and neutrophils have been found to be required for effective host protection against C.r. Yet details of the cellular sources that control neutrophil recruitment during C.r infection are poorly understood. Here we have found that C.r infection promotes neutrophil recruitment that is limited to distal colon where C.r colonizes. C.r infection induces the neutrophil-recruiting chemokines CXCL1, CXCL2 and CXCL5 in distal colon. Early in infection (D4) intestinal epithelial cells serve as the dominant source of CXCL1/2/5. However, later in infection (D8) neutrophils also provide CXCL1 and CXCL2, but fail to produce CXCL5, production of which is sustained in superficial epithelial cells. These findings support a model in which production of neutrophil-attracting chemokines is initiated in the infected epithelium and is amplified in a feedforward manner by incoming neutrophils. However, the unique production of CXCL5 by epithelial cells may be essential to recruit neutrophils to the mucosal surface, which C.r colonizes during infection.
Novel fluorochrome development enables 34-color high-content flow cytometry
Authors: Seong Yekyung , Nguyen Denny , Thakur Archana , Harding Fiona A , Nguyen Andrew 
Published: January 2023

Abstract
Flow cytometry is an essential technology to identify and quantify cell populations. Here we examined the development of new fluorochromes that could enhance flow cytometry’s capability. The latest spectral flow cytometers have 3 excitation lasers (405nm, 488nm, and 640nm) and incorporate the Avalanche Diodes Photodetector technology, which demonstrates significant improvement in sensitivity for the fluorescent emission signal longer than 800nm. However, there is no commercially available fluorochrome which could be excited by the above lasers and has peak emission signal beyond 800nm. To address the gap in technology, here we engineered 6 new fluorochromes: PE-750, PE-810, PE-830 for blue laser and APC-750, APC-810, APC-830 for red laser. These were created by covalently linking a protein donor dye with an organic small molecule acceptor dye. Then, the fluorochromes were conjugated with antibodies using Click chemistry, which is a simple, robust reaction with high-yield and high-reaction sensitivity. Also, we demonstrated long-term stability at −20°C with protein stabilizing cocktails. Most importantly, in order to show the utility of these novel fluorochromes, we created a 34-color flow cytometry panel to measure broad human immune function with high sensitivity on a 3-laser Aurora. The additional UV laser upgrade will increase the instrument capacity to more than 40 color.
This panel will be applied to identify the expression patterns of many different receptors involved in trafficking on multiple different cell types from dissociated tumors and blood samples. In conclusion, this high-content flow cytometry panel will improve analysis and diagnosis in Immuno-oncology and facilitate innovation in biomarker discovery.
Demonstration of High Gain mode in combination with Imaging Flow Cytometry for improved EV analysis
Authors: Pugsley Haley Renee , Kong Raymond K 
Published: January 2023

Abstract
Extracellular vesicles (EVs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. In recent years, the importance of EVs has become apparent, as they are key mediators of intercellular communication. However, quantifying and characterizing EVs in a reproducible and reliable manner is challenging due to their small size—exosomes range from 30 to 100 nm in diameter. It is well-known that flow cytometers were originally designed to measure and detect cells, and due to the quantitative power flow cytometry offers, there has been a push to quantify and characterize EVs using flow cytometric methods. However, these systems have not been designed to measure objects smaller than a cell. Here, we describe the use of High Gain mode on the Amnis® ImageStream® Imaging Flow Cytometer to address the challenges of measuring small particles. In this new High Gain mode, the charge-coupled device (CCD)-camera is manually adjusted to higher gain settings, increasing the signal obtained from the EV. Object thresholds and masking have also been adjusted to better identify and detect small particles. Preliminary results using murine leukemia virus-sfGFP reference particles have shown up to a 5-fold increase in the number of GFP-positive objects collected in High Gain mode, when compared to standard gain on the ImageStream® System. In this study, we demonstrate improved small particle detection, including EVs, using this new High Gain mode.
Deep characterization of in vitro chronically stimulated T cells via single-cell multiomic analysis
Authors: Corselli Mirko , Saksena Suraj , Nakamoto Margaret , Lomas Woodrow E , Taylor Ian , Chattopadhyay Pratip K 
Published: January 2023

Abstract
To generate enough cells for viable adoptive cell therapy, cells must be robustly stimulated, which raises the risk of inducing T-cell exhaustion and reducing therapeutic efficacy. To comprehensively characterize in vitro chronically expanded human T cells, we performed single-cell multiomic analysis using BD® AbSeq and BD Rhapsody™ Single-Cell Analysis system for simultaneous measurement of the expression of 38 proteins and 399 genes. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states. T-cell activation induced by anti-CD3/CD28 beads and recombinant human IL-2 led to downregulation of naïve-associated markers and upregulation of effector molecules, proliferation regulators, co-inhibitory and co-stimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation defined by markers temporarily expressed upon 3 days of stimulation, markers constitutively expressed throughout chronic activation, and markers uniquely up-regulated upon 14 days of stimulation. These data indicate heterogeneity and plasticity of chronically stimulated T cells in response to different kinetics of activation. We demonstrate the power of a single-cell multiomic approach to comprehensively characterize T cells and to precisely monitor changes in differentiation, activation and exhaustion signatures in response to different activation protocols.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
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A novel radio-pharmaceutical to image activation of the innate immune system in vivo
Authors: Gammon Seth Thomas , Pisaneschi Federica , Paolillo Vincenzo , Qureshy Sarah , Piwnica-Worms David 
Published: January 2023

Abstract
Dysregulation of innate immunity contributes to the pathophysiology of many diseases, including obesity, rheumatoid arthritis and diabetes. Dysregulated production of reactive oxygen (ROS) and nitrogen species (RNS) by the innate immune system act both malevolently and benevolently through tissue damage and T-cell suppression. Optical reporters such as DCFDA and luminol/L-012 are routinely utilized to monitor ROS/RNS. 4-chloro-1-napthol (4CN) has been used to identify innate immune cells, and 1-napthol (N) covalently binds to activated human neutrophils through Nox2 and MPO-dependent mechanisms. However, these compounds are not suitable for deep tissue imaging in vivo. Under-standing this complex interplay in humans may benefit from deep tissue imaging of ROS/RNS by positron emission tomography (PET).
Herein we introduce [18F]4FN, a bioisostere of N and 4CN, as a novel PET imaging agent, and validate binding both in vitro and in vivo. In all-trans-retinoic acid-differentiated HL-60 cells, [18F]4FN showed a robust 4.7 +/− 0.5 fold increase in binding to PMA-activated cells vs control cells (SEM n = 4, p = 0.01). Binding was &gt; 99% inhibited by treatment with the peroxidase inhibitor 4-ABAH and the Nox2 inhibitor DPI (p &lt; 0.01, each). [18F]4FN was selectively trapped in inflamed mouse tissues in vivo upon activation by PMA or LPS, robust to injection route (ip vs iv) and strain (C57Bl6/N and Balbc/N). These data quantitatively co-correlated with a gold standard bioluminescent reporter, L-012 (r = 0.94, p = 0.0004), and immune infiltration was cross validated by H&E staining ex vivo. [18F]4FN showed strong promise in pre-clinical models and translational steps toward clinical PET imaging is underway.
Adarza’s ZIVA multiplex protein detection platform offers format for ultra-high sensitivity
Authors: Schmuke Jon J , Haley Jeaneen , Wente-Roth Traci 
Published: January 2023

Abstract
We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon chip, to create a multiplex antibody array for the detection of both low and highly abundant targets. ZIVA is a new multiplex protein detection platform that leverages AIR™ technology. Because of the label-free properties, sensitivity can be “fine-tuned” by leveraging a variety of mass enhancing options. We have previously demonstrated assays that respond in the nanogram to picogram/ml sensitivity using one format for enhancement. Herein, we demonstrate a newly developed mass enhancement option that increases sensitivity from very low pg/ml to mid-femtogram/ml levels. A custom designed biosensor with markers for inflammation and other custom needs was used to demonstrate this ultra-high sensitivity format in a multiplex array. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the ZIVA instrument, the technology has been designed for the ultimate user-experience.
Protein- and sequencing-based massively parallel single-cell approaches to immune profiling asthma-specific cell subtypes and related gene expression biomarkers
Authors: Ji Xuhuai , Acharya Swati , Goncharov Igor , Lyu Shu-Chen , Miranda Molly , Padalon-Brauch Gilly , Nadeau Kari Christine , Maecker Holden T. 
Published: January 2023

Abstract
We implemented the BD Rhapsody Single-Cell Analysis System to assess cellular heterogeneity present in twins by capturing and sequencing over 40000 single cells across 10 samples from 5 twins patients with asthma or without asthma using oligos-conjugated antibodies and the whole blood immune targeted gene panel that utilizes multiplex PCR for detection of over 500 genes for immune cells subtyping, immunophenotyping, cell-type identification and markers correlated with stimulations. A combinatorial library of beads bearing cell- and molecular-barcoding capture probes is used to uniquely label transcripts and reconstruct the digital gene expression profile of thousands of individual cells in a single experiment without the need for robotics or automation. We demonstrate the ability of the BD Rhapsody targeted panel to distinguish six different cell types and 18 distinct gene-expression biomarkers, including CLEC4E, CSF2, IL1RN, TPSAB1, CCL19, CCL22, CXCL2, LAMP3, SPP1, and VMO etc.. The HLA-DRA, HLA-DQB1, SPP1, and IL1RN had allelic polymorphism related to asthma. The BD Rhapsody targeted panel yields more sequencing depth with much fewer (&lt;2%) sequencing reads per cell. The measurement of specific proteins and transcripts in individual cells is critical for understanding the role of cellular diversity in development, health, and disease.
Eukaryotic Translation Initiation Factor 4E (eIF4E) is Required for Development of T Follicular Helper Cells and Pathogenesis of Autoimmune Encephalitis
Authors: Patel Preeyam S , Walters Beth , Orlen Margo , Volkova Angelina , Ruggles Kelly , Park Christopher , Schneider Robert 
Published: January 2023

Abstract
Multiple sclerosis (MS) is a neuroinflammatory disorder resulting from infiltration of T cells into the central nervous system (CNS) and demyelination of neurons. Since T follicular helper (TFH) cells are associated with MS relapse, their selective inhibition could be an ideal therapeutic. TFH cells require transcription factor BCL6 and active mTORC1/2 for development. When mTORC is active, cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) initiates translation of selective mRNAs. However, the requirement of eIF4E for translation of mRNAs necessary for TFH cell development has not been investigated. Disruption of eIF4E binding to the 5′ mRNA cap with drug 4EGI-1 inhibits TFH and germinal center (GC) B cell development while having no effect on differentiation and effector function of TH1, TH2, TH17, or Tregs. Silencing of eIF4E in only CD4 T cells is sufficient to inhibit their formation of TFH cells. We used polysome profiling to determine which mRNAs are selectively translated by eIF4E and identified salient programs regulated by transcription (BCL6, NFAT) and costimulation (CD28, SLAM). eIF4E is required for translation of BCL6 in human lymph node TFH and GC B cells. Administration of 4EGI-1 during experimental autoimmune encephalitis (EAE) results in significantly decreased infiltration of CD4 T cells in the CNS, demyelination, and clinical score. Further, 4EGI-1 treatment following initiation of symptoms results in rapid improvement of symptoms and partial remission earlier than vehicle-treated animals. Thus, eIF4E is required for differentiation of TFH cells and pathogenesis of autoimmune encephalitis, and 4EGI-1 represents a potential therapeutic.