bluuebunny/crossref_metadata_2025_split huggingface dataset:
A pathogen-free flow cytometry based opsonophagocytosis assay protocol to quantify antibody-mediated phagocytosis
Authors: Badten Alexander Jesse , Felgner Jiin , Pone Egest J , Albin Tyler J , Fellon Corey , Kang Karen , Felgner Philip L , Burkhardt Amanda M 
Published: January 2023

Abstract
An important function of antibodies is their ability to mark cells for phagocytosis and subsequent degradation in a process known as opsonization. When developing new vaccine candidates, it is critical to determine whether the antigen specific antibodies elicited in response to immunization have this function. Due to the high containment working constraints of several important human pathogens, it is not always possible to directly study this property using the pathogen of interest. Therefore, we sought to develop a pathogen-free, flow cytometry based assay to quantify the opsonizing activity of antibodies elicited in response to immunization. We studied this using vaccine candidates against Coxiella burnetii, the causative agent of Q-fever that is a potential bioterrorism agent. In our assay, fluorescent polystyrene beads were conjugated to C. burnetii proteins then incubated with the antibody-containing sera. The antibody-coated beads were incubated with macrophages and phagocytosis of the fluorescent beads was assayed by flow cytometry. This assay allows for rapid quantification of the opsonizing capacity of antigen specific antibodies elicited in response to immunization.
Cis-proteomics reveals novel transcriptional regulators of Treg development and function
Authors: Feng Yongqiang , Zong Xinying 
Published: January 2023

Abstract
The immune suppressive function of regulatory T (Treg) cells is governed by the Foxp3-depenent transcriptional programs that remain unclear, which constraints effective manipulation of Treg behaviors for the treatment of many autoimmune diseases and cancer. To overcome the technical barriers for addressing this issue, we developed a new method to reveal the protein components of the cis-regulatory elements (cis-proteome) in Treg cells defined by Foxp3 binding, because the interaction among these DNA elements and their associated proteins ultimately determine Treg development and function. Specifically, we projected the spatial information (PSI) of Foxp3 onto its adjacent chromatins and nuclear factors with a proximity ligation reaction and identified the labeled DNA-protein complexes with chromatin immunoprecipitation (ChIP) and quantitative mass spectrometry-based proteomics.
Our Foxp3 PSI reproducibly captured majority of the open chromatins and active enhancers and promoters, representing the Treg cis-regulatory hubs, and identified 1500 proteins including many known Foxp3 interactors, crucial regulators of Treg function, and numerous novel nuclear proteins that encompass all the key steps of gene regulation. Among them 124 are highly variable in their expression levels in a cell type- or activity-dependent manner. Using a CRISPR/Cas9 approach we identified more than 50 novel nuclear factors specifically required for the induction or maintenance of Foxp3 expression.
Our study thereby offers a broadly applicable strategy to investigate the proteins involved in Treg differentiation and function and will produce many mechanistic insights into the transcriptional basis of Treg-mediated immune tolerance.
Single cell whole transcriptome analysis of disease cells to generate a targeted RNA-sequencing gene panel for the simultaneous analysis of targeted mRNA and protein
Authors: Bansal Nidhanjali , Song Hye-Won , Sa Silin , Lomas Woodrow E , Baracho Gisele V , Taylor Ian , Widmann Stephanie , Mortimer Stefanie 
Published: January 2023

Abstract
Single cell RNA-sequencing (scRNA-seq) is a powerful tool for understanding the sample heterogeneity of individual cells. Many methods rely on whole transcriptome analysis (WTA) to get a snapshot of the entire cellular landscape. Although WTA analysis can be used to discover novel biomarkers, this technique can be expensive. To enable scaling of experiments, WTA data can be mined to design targeted gene panels. To showcase this, we used single cell sequencing to examine thousands of B cells isolated from the bone marrow and peripheral blood of chronic lymphocytic leukemia (CLL) and healthy donors. B cells that had been sorted using the BD FACSMelody™ cell sorter were multiplexed using the BD™ Human Single-Cell Multiplexing Kit and pooled before being processed on the BD Rhapsody™ system, thereby minimizing batch effects. The resulting data was mined to design a panel of differentially expressed genes between CLL and healthy B cells that could be used for subsequent CLL phenotyping. By combining this panel with the BD Rhapsody™ Immune Response Panel (together comprising ~500 mRNAs), along with 36 DNA-barcoded BD™ AbSeq antibodies, we were able to simultaneously analyze mRNA and protein targets from a new subset of CLL and healthy B cells for additional high-resolution analysis. This study showcases the power of using WTA data to design specific gene panels that can be used alone or in combination with existing targeted panels for routine and cost-effective transcriptional profiling at a single cell level.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD, the BD Logo, FACSMelody, and Rhapsody are trademarks of Becton, Dickinson and Company. © 2019 BD and its subsidiaries. All rights reserved.
Evaluation of Cytometer Sensitivity and Stability using Automated Analysis
Authors: Bradford Jolene A , Meskas Justin , Sardina Patricia 
Published: January 2023

Abstract
Flow cytometers are standard tools of the immunologist, providing simultaneous measurement of many targets. The performance of a flow cytometer may vary between instruments, or over time within the same instrument. Evaluation of cytometer performance is essential to ensure results are consistent over time within the same instrument for longitudinal studies and day-to-day data consistency, and across multiple instruments when more than one cytometer is being utilized. Basic capabilities of each fluorescent detector can be estimated using the Q & B calculations, where Q is detection efficiency and B is background. Precision of measurements and minimum detectable dye can be derived from Q & B measurements, to evaluate sensitivity and stability.
In this study hard-dyed multi-level, multi-dye beads were collected in three different identically configured cytometers over 11 days. Each day data was collected in 14 different fluorescent detectors at six flow rates. An automated approach to data analysis was utilized to provide unbiased evaluation. All data files were first evaluated with flowCut to check for spurious events. Using automated gating, beads were then identified using Forward Scatter vs. Side Scatter, and flowQB was used to calculate Q & B. This data was used to evaluate intra-instrument and inter-instrument sensitivity and stability.
Results show the minimum amount of detectable dye and precision of its measurement are robust across time and different cytometers, though decreased at the highest flow rates of ≥ 500 μL/min. Using an automated unbiased approach for determining Q & B demonstrates cytometer sensitivity and consistency over time, between instruments, and across flow rates.
Developing a novel multiplexed high throughput flow cytometry based immune assay to screen kinase modulators of primary T cell activation
Authors: O’Rourke John , Gomez-Donart Andrea , Weldon Caroline , Liu Zhaoping 
Published: January 2023

Abstract
Modulating TCR engagement and signaling using biologics, small molecules or genetic engineering is highly relevant to many therapeutic areas. TCR signal initiation is mediated by cytosolic tyrosine kinases, leading to signal amplification through a network of serine-threonine kinases. Genetic defects, mutations and other mechanisms leading to increased kinase activity and enhanced T cell activation (TCA) is involved in many autoimmune pathologies, making kinases attractive targets for the direct inhibition of TCA and treatment of autoimmune disease.
The development of drugs and therapies regulating TCR activity require assays to profile T cell function and cell health. To address this need, we developed a novel multiplexed TCA assay based on high throughput flow cytometry technology with advanced computational algorithms for data analysis. The assay simultaneously measures T cell phenotype, time-dependent expression of T cell activation markers, cell proliferation and viability and quantitates secreted cytokines from a single 10 ml sample using a 96 or 384-well plate format.
To illustrate the value of the TCA assay in drug discovery, we performed a phenotypic screen using a 152 kinase inhibitor (KI) small molecule library for their ability to inhibit human primary TCA. Using the Profile Mapping algorithm in our ForeCyt software, we integrated 11 TCA metrics to identify multiple inhibition pathways. The Profile Map tool was used to group KI based on their inhibition phenotypes, irrespective of their known targets. These results illustrate the use of Intellicyt’s technology to deliver solutions for drug and biologics discovery.
Type II NKT cells directed immune regulatory mechanism(s) control spontaneous lupus nephritis in mice
Authors: Maricic Igor , Halder Ramesh , Marrero Idania , Kono Dwight H , Kumar Vipin 
Published: January 2023

Abstract
NKT cells recognize lipid antigens presented by CD1d molecules and can be categorized as iNKT cells and type II NKT cells. Type II NKT cells recognize sulfatide or lysophosphatidylcholine (LPC), and their activation results in cross-regulation of iNKT cells, tolerization of DC and control of inflammatory diseases. iNKT cells have been shown to play a pathogenic role in (NZB x NZW)F1 or BWF1 mice, a prototype for human SLE. Interestingly, anti-glycosphingolipid antibody responses have been found in lupus patients. Since sulfatide is enriched in kidney glomeruli, we have examined the role of type II NKT cells in the spontaneous development of lupus in BWF1 mice. We found that both iNKT (αGalCer/CD1d-tetramer+) and type II (sulfatide/CD1d-tetramer+) NKT cells accumulate in kidney tissues with progression of disease in BWF1 mice. Notably, iNKT cells are also activated in SLE patients and secrete the pro-inflammatory cytokine IFNg. Sulfatide-mediated activation of type II NKT cells leads to a significant inhibition of nephritis in BWF1 mice. Importantly, a clinically relevant structural analog of LPC, Miltefosine, also binds to CD1d, activates type II NKT cells and upon oral administration induces inhibition of proteinuria, anti-DNA Abs as well as infiltration of CD4+/CD8+T cells and B cells into kidney of BWF1 mice. Interestingly, the phenotype and frequency of type I interferon-secreting pDC is also altered in kidney of mice treated with Miltefosine. These studies suggest a key role for a type II NKT cell-based immune regulatory mechanism in the control of lupus and, since the CD1d-dependent pathway is highly conserved from mice to human, they have important implications for repurposing a drug for immunotherapeutic for lupus.
A nanoparticle-based approach targeting ion channels for the treatment of Lupus nephritis
Authors: Chimote Ameet A , Khodoun Marat , Duncan Heather J , Kant Shashi K , Conforti Laura 
Published: January 2023

Abstract
Lupus nephritis (LN) occurs in up to 60% of patients with systemic lupus erythematosus (SLE) and it remains the predominant cause of morbidity and mortality. Current available therapies for SLE have serious side effects and limited efficacy. Our research aims to develop a novel therapy for SLE with improved efficacy and minimal side effects. SLE is in large part driven by hyperactive T cells, which present with multiple defects including increased INFγ, chemokine receptors and CD40L that exacerbate tissue infiltration, inflammation and B cell stimulation. Kv1.3 channels regulate the Ca2+ signaling necessary for these effector functions. We have developed lipid nanoparticles that selectively target memory T (TM) cells and reduce their hyper-functionality by downregulating Kv1.3 via siRNAs (Kv1.3-NPs). Our in-vitro data showed that Kv1.3-NPs suppressed Ca2+ signaling, CD40L expression and IFNγ release selectively in TM cells. To demonstrate therapeutic efficacy of the Kv1.3-NPs we have developed a humanized mouse model of LN by engrafting peripheral blood mononuclear cells (PBMCs) from LN patients into immune-deficient NSG mice. Kidneys of these mice were highly infiltrated with human CD3+ T cells and these cells produced elevated IFNγ. Furthermore, these mice demonstrated a lower survival compared to control mice engrafted with PBMCs from healthy donors. Lastly, we tested the efficacy of the Kv1.3-NPs. We found that LN mice engrafted with PBMCs pretreated in-vitro with Kv1.3-NPs survived longer than mice engrafted with PBMCs pretreated with scramble RNA-NPs. We also found that Kv1.3-NPs were selectively delivered in-vivo into TM cells of humanized mice. Overall, these data support the potential therapeutic value of Kv1.3-NPs.
Investigational Treatment of Rheumatoid Arthritis with a Vibrotactile Device Applied to the External Ear
Authors: Imperato Gavin H. , Addorisio Meghan E. , Pavlov Valentin A. , Yang Huan , Diamond Betty , Tracey Kevin J. , Chavan Sangeeta S. 
Published: January 2023

Abstract
Rheumatoid arthritis (RA) is a debilitating polyarthritis characterized by joint tissue inflammation. Here, we assessed the effect of applying a vibrotactile device to the external ear on inflammatory responses in healthy subjects, as well as its effect on disease activity in patients with RA. We enrolled 19 healthy subjects in a randomized cross-over study, delivered vibrotactile treatment at either the external ear or gastrocnemius, and performed ex vivo whole blood assays. Vibrotactile treatment at the external ear significantly reduced TNF, IL-1β, and IL-6 levels compared to pre-treatment baseline (TNF p&lt;0.05, IL-1β p&lt;0.005, IL-6 p&lt;0.005), whereas treatment at the gastrocnemius did not attenuate inflammatory responses. Vibrotactile treatment at the external ear inhibited TNF by 80% (mean ± SD: pre-treatment = 4541 ± 2721 pg/ml vs. post-treatment = 3624 ± 2810 pg/ml), IL-6 by 73% (mean ± SD: pre-treatment = 5979 ± 2094 pg/ml vs post-treatment = 4342 ± 2600 pg/ml), and IL-1β by 50% (mean ± SD: pre-treatment = 1527 ± 1429 pg/ml vs post-treatment = 765 ± 968 pg/ml) as compared to pre-treatment baseline levels. 9 patients with RA were enrolled in a prospective interventional study. Vibrotactile treatment at the external ear significantly decreased DAS28-CRP scores two days post-treatment (DAS28-CRP score mean ± SD: pre-treatment = 3.4 ± 1.4 [1.27–5.67] vs. post-treatment = 2.7 ± 0.9 [1.24–4.37], p&lt;0.005). DAS28-CRP scores remained significantly reduced 7 days post-treatment (DAS28-CRP score mean ± SD: 7 days post-treatment = 2.4 ± 0.7 [1.21–3.68]; p&lt;0.005). These data demonstrate that application of a vibrotactile device to the external ear attenuates systemic inflammatory responses in both healthy subjects and patients with RA.
Restraining neuroinflammation during antigen delivery as an immunosuppressive approach to prevent and treat contact dermatitis
Authors: Bandyopadhyay Mohna , Morelli Adrian E , Erdos Geza , Sumpter Tina L , Tkacheva Olga A , Shufesky William J , Falo Louis D , Larregina Adriana T 
Published: January 2023

Abstract
Neuroinflammation promotes the initiation and sustains chronic inflammatory disorders. Substance-P, released by sensory neurons, is the prototype neuropeptide that signals via the neurokinin 1 receptor (NK1R) to enhance cellular immunity. We observed that NK1RKO or SPKO mice do not develop contact dermatitis (CD). Hence, we hypothesized that limiting neuroinflammation during Ag entrance induces an immune-suppressive environment to prevent T cell priming and eliminate memory T cells that cause CD. We co-delivered OVA or 2,4-dinitrocholorobencene (DNCB) and two different NK1R antagonists during sensitization of a DTH reaction induced to C57/BL6 mice reconstituted or not with OTI and OTII cells. For efficient skin delivery, we generated microneedle arrays loaded with OVA or DNCB and NK1R antagonists. We demonstrate that our approach prevents the innate and adaptive immunity accounting for the initiation of CD and mitigates pre-existing pathogenic memory T cells that cause local or systemic CD relapses. Mechanistic studies demonstrate that NK1R antagonists suppress the release of pro-inflammatory cytokines in the skin, promote the death of activated CD4 Th1 and CD8 T-cells, and generate Tregs in the skin draining lymph nodes. Our data demonstrates that controlling neuroinflammation during Ag entrance prevents the generation of pathogenic effector and memory T cells accounting for the initiation and relapses of chronic inflammatory skin disorders like CD. NIH R01 AR068249 and AR071277 to ATL and LDF.
De novo DNA methylation programs regulate T cell exhaustion and limit T cell-based immunotherapies
Authors: Youngblood Ben A , Ghoneim Hazem E. , Abdelsamed Hossam Aly , Moustaki Ardiana , Fan Yiping , Crawford Jeremy , Thomas Paul Glyndwr , Stewart Elizabeth , Federico Sara 
Published: January 2023

Abstract
T cell-based immunotherapies have emerged as one of the most promising frontiers in the fight against cancer and chronic infections. However, it has become clear that prolonged exposure of T cells to their cognate antigen drives them toward a terminally differentiated state, limiting their capacity to mount an effector response. The commitment of T cells to this “exhausted” fate is currently a major barrier in the advancement of T cell immunotherapy efforts. To better understand mechanisms that reinforce the T cell exhaustion gene expression program, we investigated the role of de novo epigenetic programming in establishing exhausted T cells that are non-responsive during immune checkpoint blockade (ICB) therapy. Using mouse models of tumor and chronic viral infection, we observed that genetic deletion of the de novo DNA methyltransferase, Dnmt3a, in antigen-specific T cells that are chronically stimulated allowed them to remain highly functional despite expressing high levels of the checkpoint inhibitory receptor PD-1. Furthermore, PD-1 blockade treatment resulted in massive expansion of PD-1+ Dnmt3a-deficient antigen-specific T cells. Building upon the findings from our murine studies, we have generated whole-genome DNA methylation profiles of human CD8 T cells from a wide array of differentiation states, and established an epigenetic-based human T cell multipotency index. Using this index, we report that tumor-associated PD-1hi CD8 T cells from pediatric solid tumors acquire an epigenetically reinforced terminal differentiation program. Collectively, these data establish Dnmt3a-mediated de novo DNA methylation programming as a key factor in limiting CD8 T cell-based immunotherapuetic approaches.
Ex Vivo Phenotyping and Potency Monitoring of CD19 CAR T Cells With a combined Flow Cytometry and Impedance-based Real-Time Cell Analysis Workflow
Authors: Lei Ming , Ye Peifang , Lu Yan , Ji Xiaoping , Wang Andy , Li Nancy , Wang Xiaobo 
Published: January 2023

Abstract
Cancer immunotherapy is increasingly utilized for cancer treatment. CARs (Chimeric antigen receptors) targeting CD19 have produced strong antitumor responses in hematologic malignancies, but tumor regression has seldom occurred when CARs targeting other antigens. The state of T-cell differentiation and T cell exhaustion influences the engraftment and persistence of CAR-expressing T cells following adoptive transfer.
Here we report the use of a combined impendence-based Real-Time Cell Analysis (RTCA) and flow cytometry cell analysis workflow for ex vivo cytolytic potency monitoring of CD19 CAR T cell, as well as the examination of phenotypic and functional responses to antigen exposure over time. CD19 CAR T cell killing of HEK293 CD19 with different E:T ratio was monitored by RTCA system. Cytokine productions were measured by NovoCyte® Quanteon™ flow cytometer after 24 hrs. Co-incubation with HEK293 CD19, phenotypic and functional responses of CD19 CAR T cells towards exposure of different antigen levels were systematically examined over time. Both assays reveal that the CD19 CAR T cells show high dose-dependent killing activities against HEK293 CD19 but not against HEK293. Using the multi-color Flow Cytometry immunophenotyping, CD4 dim and CD8 dim populations are identified as effective killing cells which are enriched in central memory (CD45RA+CCR7−) cells while normal CD4+ or CD8+ T cells consist of predominant naïve-like (CD45RA+CCR7+) T cells after co-incubation with HEK293 CD19 cells. Exhaustion marker (PD-1, LAG-3, TIM-3) are overexpressed in normal CD4+ and CD8+ populations but not in CD4 dim and CD8 dim populations with CD19 antigen exposure. After antigen exposure, T cell activation levels change significantly.
CAR+ T cell anti-tumor efficacy revealed by multi-dimensional single-cell profiling
Authors: An Xingyue , Romain Gabrielle , Martinez-Paniagua Melisa , Bandey Irfan N , Adolacion Jay R T , Fathi Mohsen , Liadi Ivan , Sadeghi Fatemeh , Mahendra Ankit , Amritkar Amit , Roysam Badrinath , Singh Harjeet , Cooper Laurence J , Varadarajan Navin 
Published: January 2023

Abstract
T cells engineered to express chimeric antigen receptor (CAR) targeting CD19 have shown promising clinical responses in patients with certain hematologic malignancies, however, it is desirable to be able to enrich cells with enhanced anti-tumor efficacy prior to infusion. We utilized a suite of high-throughput technologies with single-cell resolution, including Timelapse Imaging Microscopy In Nanowell Grids (TIMING) that integrates cytokine profiling to reveal that persistent motility of CD19- specific CAR T cells is correlated to desirable polyfunctionality (elimination of tumor cells and cytokine secretion), contributing to anti-tumor effects. We implemented a marker-free Boyden chamber-based method to enrich CAR+ T cells with persistent motility (motile cell). Integration of transcriptomic profiling, immune phenotyping and metabolism demonstrated that motile cells are more naïve-like with higher oxidative metabolism and spare respiratory capacity. Our result also revealed that the master metabolic regulator AMP kinase (AMPK) is required for CAR+ T cells with high motility. We used a xenograft leukemia mouse model (CD19+ NALM-6) and validated that the motile cells have enhanced persistence and superior anti-cancer effect in vivo compared to the parental un-sorted population. Collectively, our multi-dimensional results demonstrated that persistent motility is a selectable biomarker of expanded CAR+ T cell bioactivity.