bluuebunny/crossref_metadata_2025_split huggingface dataset:
Generation of a novel GATA3 reporter and inducible deletion system to study GATA3hi Th2 and ILC2 cells in vivo
Authors: Gurram Rama Krishna , Liu Chengyu , Zhu Jinfang (Jeff) 
Published: January 2023

Abstract
GATA3 is an important transcription factor for the development of CD4 T cells and innate lymphoid cells (ILCs); it is also essential for the differentiation and function of Th2 cells and ILC2s. To understand the physiological role of GATA3 in these cells particularly in vivo, we have generated a novel GATA3 reporter mouse strain by inserting the ZsGreen-T2A cassette in front of the translation initiation site of the Gata3 allele already carrying two loxp sites which allow inducible Gata3 deletion when it is crossed to the CreERT2 transgenic mouse strain. Our initial experiments with this mouse line showed that the reporter expression faithfully reflects the GATA3 protein expression in both in vitro differentiated Th subsets and in vivo isolated T cells and ILCs. Furthermore, the reporter expression was still observed even when the Gata3 exon 4 (encoding zinc fingers) was deleted by tamoxifen treatment. Thus, this novel mouse model will allow us to separate GATA3-deficient “Th2” cells and “ILC2s” during in vivo immune responses. We are now using this reporter system to explore the role of GATA3 in various cells types generated in vivo by comparing GATA3-sufficient and GATA3-deficient reporter positive cells.
Using immune cell type-specific gene backgrounds for refined GO enrichment analysis: an example from CD4 T cell transcriptomics of TB-infected individuals
Authors: Pomaznoy Mikhail , Ha Brendan , Peters Bjoern 
Published: January 2023

Abstract
Gene Set Enrichment Analysis (GSEA) and its special case of GO enrichment analysis is a common step in biologically interpretation of gene lists resulting from –omics experiments. A known but under-appreciated aspect of any GSEA is the use of a background gene list, e.g. by discarding genes generally not expressed in the investigated cell type. We recently developed a web-application for interactive GO analysis of gene/protein signatures named GOnet (https://tools.dice-database.org/GOnet/). Within its framework we utilized DICE-DB (https://dice-database.org/) expression data of 15 blood cell types to allow analyzing gene signatures versus genes expressed in these cell types. To validate our approach, we applied GOnet to analyze a 74-gene transcriptomic signature of latent TB infection obtained by RNA-seq of CD4 memory T cells (Burel et al, 2018). Limiting the analysis to the background of genes expressed in CD4 T cells introduces significant differences in GO enrichment analysis results. Firstly, the category of “regulation of immunological synapse formation” is no longer enriched. As immunological synapse genes are very specific for T cells, without background correction this category can very easily seem ‘enriched’. Secondly, a more specific background renders a category of “positive regulation of MAPK cascade” significant. The latter category highlights important regulators contained in the signature. Overall we believe that using baseline gene sets based on immune cell types expression profiles is an important advancement to regular GO enrichment analysis in immunology studies, and by making relevant backgrounds readily available in GOnet, applying them becomes easier for immunologists.
Detection of small particles by flow cytometry: Analysis of small particle beads and extracellular vesicles
Authors: Jachimowicz Lauren A , Ye Peifang , Lu Yan , Guenther Garret , Nancy LI 
Published: January 2023

Abstract
The importance of cell-derived small particles has recently been recognized in multiple biological processes and is an increasing topic of investigation in biomedical research. Extracellular vesicles (EV) are cell-derived membranous vesicles that include exosomes, microvesicles, and apoptotic bodies. Small particle analysis by flow cytometry can bring much needed discoveries in small particle research, with the capability to detect rare events in significant numbers as well as measure surface protein levels. However, traditional flow cytometers were not capable of resolving them due to their small diameter and variable composition.
The aim of this investigation is to determine the capacities of the NovoCyte QuanteonTM flow cytometer to resolve small particles, using both beads and cell-derived EV. We first performed analysis of three commercially available small particle bead mixes ranging in size of 100nm-1300nm and determined the Quanteon is able to resolve beads as small as 100nm. Since beads scatter more light than similar sized membranous particles, it is important to measure biological samples to test the detection limits of the Quanteon. Therefore, HEK293 derived GFP lipoparticles were analyzed for scatter resolution and GFP expression. In addition, plasma was analyzed for purified EV with and without ADP addition, a molecule known to induce platelet activation and EV release. Next, EV from a cancer cell line, SW480, was analyzed for known EV markers CD9, CD81, and AnnexinV. The Quanteon was able to effectively analyze lipoparticles and secreted EV. In conclusion, the NovoCyte Quanteon equipped with new technologies allows better discrimination of small particles and provide increased capacity for research into EV.
Combining TruCulture and OptiMAP to Profile Human Immune Immunity
Authors: Hwang Shen-An , Eisinger Dominic , LaBrie Samuel 
Published: January 2023

Abstract
TruCulture® is a whole blood collection and culture system designed for use at clinical sites. TruCulture outperforms traditional peripheral blood mononuclear cell (PBMC) culturing methods, with improved reproducibility and higher levels of stimulation (Duffy et. al. Clinical Immunology 2017). The TruCulture proprietary culture medium ensures minimal non-specific immune activation, delivering a steady baseline and decreased variation during stimulating parameters. OptiMAP is a multiplex immunoassay panel that measures 13 cytokines of the major immune response pathways (TH1, TH2, TH17, and monocyte/macrophage). Using TruCulture and OptiMAP, we analyzed healthy donor whole blood responses to common stimulants: T cell activators, TLR agonists, and superantigens. The subjects ranged from 24 to 70 years old and consisted of 48% males and 52% females. The results indicated that healthy donors responded similarly to stimulation with anti-CD3/CD28 and Staphylococcal enterotoxin type B (SEB) in production of T cell cytokines (IFN-γ, IL-2, and IL-17). Response to SEB is positively correlated to the monocyte population in whole blood. T cell responses to SEB and CytoStim (an antibody based alternative to SEB) are similar in production of IFN-γ and IL-17, but CytoStim had a reduced stimulation of IL-2. All healthy donors responded to a TLR-4 agonist (LPS) by producing high levels of TNF-α, IL- 6, and IL-1β. LPS stimulated production of IFN-γ was similar to SEB, most likely due to bystander activation. The TLR-7 agonist (gardiquimod, GDQ) stimulated IL-10 levels at a level comparable to LPS. These data illustrate the utility of combining TruCulture and OptiMAP to investigate human immune stimulation pathways for clinical research.
Novel Flow-Based Multiplex Assay Panel for Quantifying Human Neuroinflammation Biomarkers
Authors: Sun Binggang , Chow Kay , Zhao Amy , Lehmann Jason , Jiang Weiping , Ji Shaoquan 
Published: January 2023

Abstract
The inflammation of neuronal tissues is triggered by various stimuli such as traumatic brain injury, stroke, protein aggregation and pathogens. Neuroinflammation cues such as brain injury lead to protein release and microglia activation, which in turn activate glial cells. Activated glial cells produce both pro- and anti-inflammatory factors to regulate cell migration, growth, tissue repair or cell death. Neuroinflammation is involved in acute brain injury, stroke, and many neurodegenerative diseases, such as Alzheimers’s and Parkinson’s diseases, as well as disruption and permeability of the blood-brain barrier. Measurement of the cytokines, chemokines, growth factors and other neuronal disease biomarkers is essential in understanding the mechanisms of neuronal disease progression, but this is limited by the small sample size of CSF. It is therefore desirable to be able to measure a panel of important neuroinflammation biomarkers simultaneously.
We have developed an assay panel targeting neuroinflammation biomarkers using fluorescence-encoded beads that are suitable for use on common lab flow cytometers. It allows for simultaneous quantification of the 13 key targets involved in neuroinflammation including VILIP-1, BDNF, free active TGF-β1, VEGF, IL-6, sTREM-1, β-NGF, IL-18, TNF-α, soluble RAGE, and CX3CL1 (Fractialkine). This panel has been developed with superior assay performance and specificity and has been validated by detecting expected values in biological samples including CSF samples from healthy and disease individuals. This high quality, low cost and easy to use panel will be a useful tool to the neuroinflammation research community.
Formal representation of immunology related data with ontologies
Authors: Vita Randi J , Overton James A , Cheung Kei-Hoi , Dunn Patrick , Burel Julie , Chan Syed Ahmad , Diehl Alexander D , Kleinstein Steven H , Sette Alessandro , Peters Bjoern 
Published: January 2023

Abstract
The Human Immunology Project Consortium (HIPC) is a multicenter collaboration between research centers performing large-scale human immunology studies that focus on profiling the human immune response to natural infection and vaccination. “Immune exposures” are events such as natural infection and vaccination whereby the immune system may or may not respond to the exposure. Many of the HIPC studies investigate the response of specific cell populations after a variety of immune exposures. In order to cross-compare results from the many different centers and projects, we established a standardized representation of immune exposures and cell descriptions that simplifies data collection. By standardizing how this data is collected and stored, the vast amount of data collected by these diverse projects is made significantly more useful and interoperable. The data collected by the HIPC projects is stored in the National Institute of Health, Division of Allergy, Immunology and Transplantation funded resource, the Immunology Database and Analysis Portal (ImmPort). ImmPort was modified to provide the necessary structured data fields to capture our standardized representation of immune exposures and studied cell populations with a set of data fields that primarily utilize formal ontology terms. We will discuss the process of modeling immune exposures and cell populations via ontology terms, including real life scenarios from HIPC projects, as well as collaborations with existing ontology projects in order to meet the specific needs of immunologists.
Development and application of an in vitro dendritic cell internalization assay to assess immunogenicity risk
Authors: Halimani Mahantappa , Atkuri Kondala , Goiberg Polina , You Zhiping , Steeno Greg , Tourdot Sophie , Xue Li , Lepsy Chris , Hickling Tim , King Lindsay 
Published: January 2023

Abstract
The objective of this research was to develop an in vitro dendritic cell based assay to assess if differences in internalization of biotherapeutic drugs (BioTx) and/or intra-cellular trafficking could be measured and used to help predict clinical immunogenicity risk. An unwanted immune response can adversely impact pharmacokinetics, safety and efficacy. Since immunogenicity in preclinical species is not clinically predictive there is a strong need for in vitro tools to enable BioTx drug design. Immature dendritic cells (iDCs) were selected because of their high fluid phase pinocytosis rates, ability to recycle IgGs and route immunogenic molecules to MHC-II compartments. Initial assay development evaluated iDC differentiated from a monocytic human cell line vs primary monocytes from fresh whole blood using IL4 and GM-CSF. Fluid phase internalization rates of Ovalbumin and Lucifer yellow decreased from day 3 to day 5 and CD80, CD86 and HLA-DR expression increased consistent with DC maturation. Imaging cytometry was used to measure internalization extent, rate and LAMP-1 co-localization of fluorescently conjugated ovalbumin and BioTx with known clinical immunogenicity incidence. Primary monocytes derived iDC were selected based on extent of internalization for further assay characterization in multiple donors. No target expression was observed. The extent of Adalimumab, Traztuzumab, and Utomilumab internalization was correlated with clinical immunogenicity incidence suggesting the assay may be able to detect frame work differences. Statistical power calculation of fold change set required donor numbers at 10. The assay can be used in combination with other in vitro immunogenicity assays to support BioTx design.
Imaging Cancer Immunology: Tracking Immune Cells in vivo with Magnetic Particle Imaging
Authors: Gaudet Jeff , Mansfield Jim , Goodwill Patrick 
Published: January 2023

Abstract
The rapid growth of research into immuno-oncology has fueled a need to be able to determine the location of a variety of immune cells in solid tumors. However, existing methods for that have been insufficient. Magnetic Particle Imaging (MPI) is a novel pre-clinical cellular imaging technique that can be used to non-invasively track iron-oxide tagged immune cells in vivo. In this study, we imaged the differences in biodistribution and migration of macrophages in naïve and tumor bearing mice. Imaging was performed with the MOMENTUM MPI system (Magnetic Insight Inc., CA). Macrophages were pre-loaded with tracers in vitro for tracking specific populations or loaded in situ for detection of localized inflammation. Initial macrophage biodistribution varied depending on the route of administration. Intravenous murine tail vein administration provided the best systemic delivery, but also resulted in macrophage accumulation within the liver and spleen. Direct intracranial injection increased local retention but resulted in the most tissue disruption. In the tumor bearing animals, macrophages were predominately detected in the expanding margins of the tumor. By combining the accurate quantification and specificity of MPI, information can be obtained on macrophage function and biodistribution. Other cells can also be labeled (dendritic cells, MDSCs, etc) and this information can be utilized to better understand the factors influencing immune cell migration in and around tumors.
A robust human immunophenotyping workflow using CyTOF technology coupled with Maxpar Pathsetter, an automated data analysis software
Authors: Li Stephen K.H. , Majonis Daniel , Bagwell C. Bruce , Hunsberger Benjamin C. , Baranov Vladimir , Ornatsky Olga 
Published: January 2023

Abstract
Mass cytometry, which utilizes CyTOF® technology, is a single-cell analysis platform that uses metal-tagged antibodies. An advantage of CyTOF is its ability to resolve more than 40 parameters in a single panel without the need for compensation, making mass cytometry an ideal solution for routine enumeration of immune cells. Developing a robust and highly multiplexed assay requires an optimized panel and analysis pipeline, and the analysis of complex panels is time-consuming and difficult to interpret without expertise in immunology.
The Maxpar® Direct Immune Profiling Assay™ coupled with Maxpar Pathsetter™ software is a sample-to-answer solution for human immune profiling using mass cytometry. The Maxpar Direct Immune Profiling Assay kit includes an optimized 30-marker panel contained in a lyophilized single-tube format, protocols for human whole blood and PBMC staining, and data acquisition instructions on a Helios™ system. Maxpar Pathsetter is an automated software that accepts FCS 3.0 files and reports cell counts, percentage calculations, and staining intensity. It also produces graphical elements such as histograms, dot plots, and a Cen-se’™ (t-SNE variant) graph for 36 immune cell populations.
We present analytical validation data on repeatability and reproducibility for the assay and on the precision and accuracy of Maxpar Pathsetter software. We demonstrate comparative performance between the lyophilized panel and a liquid panel of the same antibodies. Coupling Maxpar Pathsetter software with the Maxpar Direct Immune Profiling Assay reduces variability in sample preparation and subjectivity in data analysis. It allows researchers to have a streamlined solution for broad immune profiling using mass cytometry.
‘Evidence of improved transduction and preservation of relevant T cell subpopulations using different media conditions: Towards a novel CAR-T media formulation’
Authors: Martinez Becerra Francisco J , O’Connor Roddy , Ghassemi Saba , Heo David , Master Alyssa 
Published: January 2023

Abstract
The field of cell therapy is shifting away from animal origin components used in the culture or preservation of cells and replacing them with human or chemically defined components. However, this shift often leads to diminished cell proliferation and functionality due to the lack of physiologically relevant proteins. Therefore, there is an important need for generation of media supplements to optimize cell therapy. PhysiologixTM XF Human Growth Factor Concentrate (hGFC) is a cGMP, xeno-free media supplement that can be used as a replacement to supplements such as human serum. In this study, T cells were isolated from blood and cultured for 14 days in OpTmizer or X-VIVO 15 supplemented with 5% human serum or 2% PhysiologixTM XF. Population doublings and cell size were defined, as well as CD4/CD8 and memory markers (CCR7 and CD45RO) to assess phenotype. We detected an increase in naïve and central memory populations in 2% PhysiologixTM XF compared to human serum across all tested basal media. These populations have been described as highly relevant for the outcome of patients undergoing CAR-T cell therapy. We compared the transduction efficiency of a lentiviral-GFP in OpTmizer, X-VIVO 15 or RPMI, supplemented with 2% PhysiologixTM XF or 5% human serum at different multiplicities of infection (MOIs). Higher transduction efficiency was observed when using 2% PhysiologixTM XF at a high MOI of 4 and was conserved across all dilutions including a MOI of &lt;1. These results indicate that PhysiologixTM XF can be used to optimize the generation of relevant T cell subpopulations and maximize transduction efficiency even at a low MOI, thus increasing the efficiency of vector usage. These results may lead to better clinical efficacy while reducing overall costs.
Improving expression estimates of HLA alleles in NGS data using personalized genomic sequences and its effect on eQTL mapping
Authors: Crinklaw Austin , Mahajan Swapnil , Pomaznoy Mikhail , Sette Alessandro , Vijayanand Pandurangan , Peters Bjoern 
Published: January 2023

Abstract
HLA genes are the most polymorphic in the human genome. Specific genetic variants in the HLA locus are frequently associated with diseases and other traits in GWAS studies. How much of those associations are due to functional differences in the peptide binding specificity of the various HLA genes encoded in the locus, vs. how many associations are due to differences in the expression level of the HLA genes has not been systematically determined. One hurdle in assessing HLA gene expression from RNA-Seq data when using standard analysis methods is the use of a single reference genome. We compared the estimated expression of HLA genes from 15 immune cell types of 91 individuals from the DICE database, using either the reference human genome sequence, or a personalized genome sequences based on HLA typing of the donors. This analysis showed that quantifying HLA gene expression using personalized genomic sequences allows to separate SNPs in association with HLA expression that are artifacts based on differences in read-mappability from other SNPs that are truly in association with personalized expression levels.
Comparative analysis of immunoglobulin IGHD genes in vertebrates
Authors: Bhardwaj Vinnu , Safonova Yana , Franceschetti Massimo , Rao Ramesh , Pevzner Pavel 
Published: January 2023

Abstract
Finding germline immunoglobulin (Ig) V, D, and J genes is a preliminary step in many antibody studies, including development of monoclonal antibody drugs. Recent studies show that the IMGT database, the state-of-the-art database of germline Ig genes, is incomplete for many species. Moreover, modern pharmaceutical studies often involve species not present in the IMGT database (e.g., hamsters, llamas). While V and J genes are conservative and can be inferred by homology, D genes among species are highly diverse. As modern sequencing technologies (Rep-Seq) have enabled scanning millions of antibody RNAs from a single organism, we developed a tool, MINING-D, for de novo inference of functional D genes using Rep-Seq data.
We applied MINING-D to 261 publicly available Rep-Seq datasets: 160 human and 101 non-human datasets, including mouse, rat, rabbit, camel, and rhesus macaque species. Most known D genes, their known and novel variations, and some D genes not presented in the IMGT database were inferred. We further validated a significant number of the novel genes and variations using genomic data.
Thus, our tool circumvents the need of an existing database and infers the functional D genes directly from expressed Ig RNAs. It can potentially speed up the process of antibody sequencing and design of antibody drugs for species with unknown genes. Results from multiple datasets enabled comparative analysis of D genes. For example, while the same germline D genes contribute to the diversity of antibody repertoires in healthy humans, only few of them are used in specific repertoires (e.g., hepatitis) and thus can be critical for recognition of their antigens. We suggest that the obtained results will help in disease monitoring and vaccine design.
Immunophenotyping extracellular vesicles using the CellStream flow cytometer
Authors: Pugsley Haley Renee , Davidson Bryan R , Morrissey Philip 
Published: January 2023

Abstract
Extracellular vesicles are membrane derived structures that include exosomes, microvesicles and apoptotic bodies. The importance of extracellular vesicles as key mediators of intercellular communication is not well understood. Exosomes have been shown to transfer molecules between cells, potentially transmitting signals. Exosomes are released under normal physiological conditions; however, they are also believed to serve as mediators in the pathogenesis of neurological, vascular, haematological and autoimmune diseases as well as cancer. Quantifying and characterizing extracellular vesicles in a reproducible and reliable manner is challenging due to their small size (exosomes range from 30 to 100 nm in diameter). Extracellular vesicle analysis can be done using high-magnification microscopy; however, this technique has a very low throughput. Attempts to analyze extracellular vesicles using traditional PMT based flow cytometers has been hampered by the limit of detection of such small particles and their low refractive index. To overcome these limitations, we have employed the Amnis CellStream that has the advantage of high throughput flow cytometry with higher sensitivity to small particles due to the CCD based, time-delay-integration image capturing system. Data will be presented using the CellStream flow cytometer to immunophenotype extracellular vesicles derived from red blood cells and platelets.