bluuebunny/crossref_metadata_2025_split huggingface dataset:
KIR and HLA class I diversity in Pakistani and Yadav populations: a high-resolution study of immunogenetic diversity in South Asians
Authors: Kichula Katherine M , Harrison Genelle F , Nelson Cassie , Piluso Allison , Freed Brian M , Aubrey Michael , Hameed K M , Norman Paul J 
Published: February 2023

Abstract
Killer cell immunoglobulin-like receptors (KIR) modulate the activity of human NK cells by interacting with HLA class I ligands, influencing their response to pathogens, cancer and during pregnancy. Combinatorial diversity of HLA and KIR allotypes impacts NK cell functions in health and disease. South Asians are under-studied for this immunogenetic diversity, despite representing almost one quarter of the world’s population. We analyzed KIR and HLA distribution of a cohort of healthy subjects comprising 81 from Karachi in Pakistan and 71 Yadav from Tamil Nadu in Southern India. To overcome genomic region complexity, we used our high throughput NGS and bioinformatics pipeline to derive high resolution gene content and allele level KIR and HLA data. We also developed a Cas9-targeted long read method to fully sequence specific structurally divergent KIR haplotypes. We identified 110 distinct HLA class I alleles in the Karachi population and 80 in the Yadav. We observed 96% individuals to have unique HLA class I genotypes and three were shown to be homozygous across all three HLA class I genes. While overall HLA diversity is lower in the Yadav compared to the Karachi, both encode approximately two KIR ligands per HLA haplotype. We identified 125 distinct KIR alleles, including 11 characterized for the first time in this study. We identified and sequenced multiple KIR haplotypes having large-scale deletions or duplications of complete genes. Most haplotypes were observed in more than one individual, but every individual studied has a unique KIR genotype. This is the first high-resolution study of KIR and HLA class I in a South Asian population, and this will lay the foundation for further immunogenic studies of subjects from this geographic region.
Integrated approaches discovered multiple novel anti-Cas13 molecules to enhance RNA editing capacity
Authors: Wu Min , Lin Ping 
Published: February 2023

Abstract
We have used an integrated approach to finding anti-CRISPR (Acr) molecules (acrVIA1-7) to repress type VI-A CRISPR (Cas13a, an RNA targeting Cas) system. This involves a combination of transcription-translation assay (TXTL), self-targeting screen system (STSS) and guilt-by-association. We started with bioinformatics-based STSS screening technique to discover strains with self-targeting spacers by Cas13 in diverse strains containing type VI CRISPR-Cas systems, identifying the Leptotrichia wadei F0279 strain having self-targeting protospacers. Then we utilized a guilt-by-association method, a total of 11 potential Acrs candidates adjacent to aca genes have been characterized. Next, a TXTL system was applied to functionally evaluate the anti-CRISPR activities of these putative Acrs, leading to the discovery of five inhibitors of type VI-A CRISPR-Cas13a systems (AcrVIA1-5). Finally, we used second iterative method based on the known acr genes (acrIC1, acrIIA1 and acrIIC4) in strains possessing self-targets, resulting in identification of Cas13a inhibitors AcrVIA6-7, as well as AcrIB1 that inhibits type I-B CRISPR-Cas systems. These newly discovered Acrs exhibited the activities of inhibiting relevant Cas proteins in bacteria and mammalian cells, such as HEK293 cells. Hence, these newly identified acr molecules may have broad usages, such as improvement of RNA editing in Cas13-based application for human disease intervention, application in biotechnology, and study of phage-bacterium interaction.
Diagnostic approaches for early cancer detection in Pacific Youth using ctDNA technology
Authors: Sika-Paotonu Dianne , Walton Mischa , Faavae Sepola , Tagi Selafi , Guilford Parry 
Published: February 2023

Abstract
Circulating tumour DNA (ctDNA) refers to small fragments of tumour-derived DNA that escape into the bloodstream. Analysis of these ctDNA fragments can be utilized to monitor responses to treatment by providing information relating to tumour size and progression and can support clinical decision making. A potential adaptation of the ctDNA monitoring technology is proposed to support utility of ctDNA for early cancer diagnostic efforts. This would comprise a simple method for early cancer detection that would be helpful across multiple settings, including that for Pacific Peoples. Pacific Peoples living in Aotearoa/New Zealand and in the Pacific Region are disproportionately affected by specific cancer types. The potential utility of ctDNA for early cancer detection is an approach that could help reduce adverse health outcomes if successful particularly for Pacific communities. To understand more about the diagnostic approaches applicable for early cancer diagnostics for Pacific young people in living in Aotearoa/New Zealand, feedback was sought on the aspects requiring attention in the Youth setting and also in relation to the possible utility of ctDNA technology as an example. Preliminary aspects of the cancer setting identified as important for early cancer diagnosis for Pacific young people, included the need for improved accessibility, the importance of cultural context and awareness, and the need for simplicity with any technological approach used for early cancer diagnosis, such as that being proposed using ctDNA.
High-speed and high-precision fluorescence-based cell count and viability assays using the Cellaca&[trade] MX high-throughput cell counter
Authors: Bell Jordan , Huang Yongyang , Yung Sun , Qazi Henry , Hernandez Charles , Qiu Jean , Chan Leo 
Published: February 2023

Abstract
With multiple FDA-approved cell therapy products available and others in pre-clinical and clinical trials, now, more than ever, it is critical to provide fast and accurate measurements of cell concentration and viability. For certain clinical products, cell concentration is synonymous with dosage, and accurate cell viability is crucial for avoidance of harmful side effects. The complex nature of patient-derived samples makes legacy cell analysis methods such as the trypan blue dye exclusion assay difficult to perform. Nonspecific objects such as RBCs and cellular debris can significantly increase cell counting variation. Meanwhile, the need for cell-based products that are custom-tailored to each patient has increased the number of samples requiring analysis, leading to cell counting bottlenecks.
With these challenges in mind, we investigate the performance of the Cellaca™ MX. This high-throughput cell counter captures images in up to 5 FL colors for cell count and viability analysis in under 3 min for 24 samples. Comparison among multiple instruments using Jurkat cells and fluorescent beads reveals high consistency between replicate counts, between plates, and between instruments. We also compare cell counts conducted on the Cellaca™ MX with counts performed using other FL-based automated cell counting methods, as well as manual counting. In addition, we illustrate the use of the new ISO cell counting standards to evaluate and compare the performance of cell counting methods. Finally, we include real-world primary T cell linearity results over a 4-log range of concentration. The results of the experiments confirm the suitability of the Cellaca™ MX for general FL-based assays applicable to cell therapy-related workflows.
TVGN-489: A novel approach for the production of SARS-CoV-2-specific cytotoxic T lymphocytes to treat COVID-19
Authors: Flomenberg Neal , Wagner John Lyle , Sykulev Yuri , Anikeyeva Nadezhda , Huang Yanping , Sun Michael , Feng Pu , Keck Kaitlyn , O'Connor Allyson , Peedin Alexis , Karp Julie , Flomenberg Phyllis , Chang AnnaMaria , Henwood Patricia , Rising Kristin , Nikbakht Neda , Saadi Ryan , Grosso Dolores A 
Published: February 2023

Abstract
SARS-COV-2 (COVID-19) has resulted in over 2 million deaths. While vaccination is expected to decrease mortality, there remains a need for curative therapies for active infections. Uncertainties regarding the duration of post-vaccination immunity and the rapidity of mutational evolution by this virus suggest that it is unwise to rely on preventative measures alone. Dysregulation of endogenous cellular immunity has been implicated in the failure to control COVID-19 infections suggesting that allogeneic T cell therapy using nature’s curative approach to viral infections could successfully be applied to vulnerable patients. Allogeneic virus-specific cytotoxic T lymphocytes (CTLs) have a long track record of safety and efficacy in treating viral infections occurring after allogeneic stem cell transplantation. We hypothesize that this approach can be applied to the treatment of Covid-19. Covid-19 specific CTLs were produced by priming and enriching T cells from convalescent patients using COVID-19 peptides predicted or demonstrated to bind to specific HLA alleles. Utilizing the globally common HLA-A*02:01 allele as the restriction element, 7 peptides from 4 COVID-19 gene/ORF products were identified and used as a pool for serial restimulation and enrichment on a peptide pulsed, HLA-A*02:01 antigen presenting cell monolayer. This preclinical effort produced CTLs of high purity (&gt;95% CD3+/CD8+, &gt;60% positive by tetramer staining) and potency (60% lysis of peptide pulsed targets at an effector to target ratio of 3:1) in sufficient quantities for clinical application. Trials using these TVGN-489 CTLs and those produced with other SARS-COV-2 peptides and HLA restriction elements will hopefully be launched shortly.
Global profiling of O-GlcNAcylated c-Rel-dependent gene expression
Authors: Centore Joshua , Tomalka Jeffrey , de Jesus Tristan , Ramakrishnan Parameswaran 
Published: February 2023

Abstract
NF-κB c-Rel is a critical regulator of transcription in T cells. We previously showed that c-Rel is O-GlcNAcylated at a single residue, serine 350, and this posttranslational modification has a dual role in positive and negative regulation of c-Rel-dependent transcription. Here we performed a targeted RNA expression study to examine how c-Rel O-GlcNAcylation influences the transcriptional landscape of known NF-κB-dependent genes in T cells. We found groups of genes that were upregulated, downregulated or unregulated upon increased O-GlcNAcylation, and several of these genes were dependent on c-Rel serine 350 as analyzed by mutational studies. We also developed a novel site-specific antibody targeting serine 350 O-GlcNAcylated c-Rel. Our functional studies show that this novel antibody is capable of detecting hyperglycemia-induced c-Rel O-GlcNAcylation in cells isolated from diabetes patients and identify O-GlcNAcylation-dependent DNA binding of c-Rel by chromatin immunoprecipitation. Knowledge gained from the gene expression study and the use of S350 site-specific anti-O-GlcNAcylated c-Rel antibody have the potential to reveal the role of c-Rel O-GlcNAcylation in regulating T cell function in autoimmune diabetes.
Technological advancements in multiomic single cell immune profiling and analysis
Authors: Royall Ariel E , Sukovich David J , Adams Bruce A , Chi Javelin C , Reyes Daniel , Puleo Alaina R , Krishnan Sreenath , Jaffe David B , McDonnell Wyatt J , Cheung Denise , Marrs Samuel , Chen Tina , Srinavas Niranjan , Carli Natasha , Montesclaros Luz , Lau Julia , Taylor Sarah E. B. 
Published: February 2023

Abstract
Continued advances in single cell gene detection sensitivity and the ability to analyze data quickly with biological context are critical for discoveries in therapeutic research. The second version of the Chromium Single Cell Immune Profiling Solution by 10x Genomics enables highly sensitive detection of gene expression, full-length paired T-cell alpha- and beta-chain and immunoglobulin sequences, T-cell antigen specificity, and cell surface protein expression from the same single cells, allowing a comprehensive view of the immune response at the cellular level. The new workflow provides a 45% increase in the number of genes detected per cell and up to a 25% increase in the cells detected with paired full-length V(D)J receptor sequences in melanoma tumor derived cells. Using a new version of Cell Ranger (v5.0), clonotypes were grouped from &gt;30,000 cells, increasing the power to detect small clonotype expansions with fewer total cells. In addition, the new software enabled comparison of pre- and post-influenza vaccination B-cell receptor sequences from a single donor, identifying post-vaccination specific clonotypes. These technological and informatics advancements enhance a researcher’s ability to perform a broad characterization of immune cell populations at unprecedented throughput and resolution.
Immune phenotypic analysis using predesigned and lyophilized BD® AbSeq Immune Discovery Panel
Authors: Song Hye-Won , Vadrevu Samatha , Narayan Punya , Baracho Gisele , Tyznik Aaron , Martin Jody , lazaruk Katherine , Nakamoto Margaret , Mortimer Stefanie 
Published: February 2023

Abstract
High-throughput single cell RNA sequencing (scRNA-seq) has recently emerged as a powerful tool for profiling complex cell populations. While traditional scRNA-seq captures information at the transcript level, recent technology using DNA-barcoded antibodies enable multiomic analysis, examining protein alongside mRNA. Although this approach allows analyzing a large number of protein markers, the sequencing cost can escalate quickly for large panels, particularly ones with high antigen-density markers, and workflows can be long. Therefore, panels should be optimized to gain meaningful results efficiently and cost-effectively. Here, we introduce the BD® AbSeq Immune Discovery Panel (IDP), a lyophilized panel consisting of 30 antibodies against major human immune cell-types and immune response markers for analyzing multiple immune cell subsets. The BD® AbSeq IDP can be used to uncover phenotypic changes in cell subsets such as unique cell input differences or changes in the immune cell status. To test this capability, we used the IDP alongside a mRNA analysis using targeted mRNA panel and whole transcriptome assay to compare the immune phenotypes of cells from disease and in vitro stimulated samples at the single-cell level using the BD Rhapsody™ System. We propose that the BD® AbSeq IDP can be used to identify the cell types and molecular pathways impacted by the disease at the single cell level.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.
Novel violet fluorescent nanoparticles for immunophenotyping using flow cytometry
Authors: Green Angela M , Woo Deanna K , Fonseca Dora , Jordan-Dreskin Elizabeth , Li Lei 
Published: February 2023

Abstract
A novel series of fluorescent nanoparticles with violet excitation have been developed for use with flow cytometry antibodies. These exceptionally bright, photostable dyes also have narrow emission spectra, making them ideal for inclusion in high parameter multicolor panels. For this study, StarBright Violets 440, 515, 610, 670, 710 and 790 were conjugated to human B-cell, T-cell, and Treg marker monoclonal antibodies and used to immunophenotype human peripheral blood mononuclear cells (PBMCs) in an 8-color panel. Tests with this panel were conducted in different staining buffers and compared to a panel with dyes having similar spectra and the same clonality. The new StarBright multicolor panel showed better resolution of Treg populations and reliable identification of the different T- and B-cell populations across different stain buffers and conditions, which makes the StarBright Violet nanoparticles an excellent addition to the field of high parameter flow cytometry.
Using ex vivo lymph node slices to monitor changes in molecular transport after vaccination
Authors: Pompano Rebecca R , Catterton Megan A , Freeman Timothy J , Ball Alexander G 
Published: February 2023

Abstract
Understanding dynamics in the lymph node has the potential to reveal disease mechanisms and inform the design of immunotherapies. To this end, our laboratory combines ex vivo slices of murine lymph node (LN) tissue with novel microfluidic culture systems to study dynamic responses to events such as vaccination or the growth of a tumor. We have developed systems for co-culture of tissue slices from multiple organs, e.g. tumor and LN, and to deliver inflammatory stimuli to local regions of ex vivo cultures. Here, we used the latter system to assess changes in molecular diffusion through the LN after vaccination with adjuvants of varied mechanism. Mice were vaccinated with ovalbumin adjuvanted with Alum, Poly I:C, Complete Freud’s Adjuvant (CFA), or a vehicle control (saline). Lymph node slices were collected on day 4, and the diffusion coefficients of a set of non-binding dextrans were measured by using microfluidic integrated optical imaging (microIOI), a method developed previously by our laboratory (Ross et. al, Anal Chim Acta, 2019). Smaller dextrans (3 and 10 kDa), but not larger ones (70 kDa), diffused significantly faster after vaccination with Poly I:C than with Alum or saline. These data suggested an expansion of the extracellular space with an upper limit of 6.0 nm, the Stokes radius of 70 kDa dextran. Consistent with this prediction, the diffusion coefficients of an antibody immunotherapy and its isotype control (IgG, 150 kDa) remained unaltered after vaccination. Next, we will test the extent to which changes in the stromal network correlate with the changes in diffusivity after vaccination, and continue to explore the use of ex vivo LN slices to inform models of intercellular communication in this organ and immunotherapy design.
Novel Multiplexed Assays for Antibody Isotypes against SARS-CoV-2 Using Flow Cytometry
Authors: Tran Kimvan T , Clor Julie , Tyagarajan Kamala 
Published: February 2023

Abstract
The ongoing COVID-19 pandemic has prompted intense research on the role of antibodies in characterizing the emergence and sustenance of the immune response. Antibody isotype information is important in understanding disease mechanism, seroconversion, and vaccine response. While several methods provide qualitative information on SARS-CoV-2 antibodies, there is a need for quick methods with high detection sensitivity that provide relative response levels against multiple SARS-CoV-2 antigens. In this study, we show results from the development of a high-sensitivity bead-based immunoassay for flow cytometry that enables detection of IgG, IgM, or IgA antibodies in serum or plasma samples against multiple SARS-CoV-2 antigens. Antigenic targets include SARS-CoV-2 nucleoprotein, S1-subunit of the spike protein, and receptor-binding domain of the spike protein. The assay allows for quick processing, displays clear visual shifts, and provides fluorescent intensity information allowing for high detection sensitivity. Application of the assay to 30 PCR positive SARS-CoV-2 samples and 30 pre-pandemic samples provides high sensitivity and specificity for IgG analysis. IgG detection was observed in SARS-CoV-2 samples ranging from 6–75 days post-onset of symptoms in the study. IgM and IgA antibodies were also detected in several samples. The assay demonstrates good precision, with low %CVs of &lt;10% typically observed. Given that SARS-CoV-2 research encompasses both cellular and serological aspects, the ability to obtain antibody isotype information on flow cytometry platforms that enable cellular as well as bead-analysis can benefit many researchers.
For Research Use Only. Not for use in diagnostic procedures.
Extensive culturing and metagenomics enable functional and taxonomic comparison of mouse and human gut microbiotas
Authors: Pedicord Virginia Anh , Beresford-Jones Benjamin S. , Forster Samuel C. , Stares Mark D. , Notley George , Viciani Elisa , Browne Hilary P. , Kumar Nitin , Vervier Kevin , Almeida Alexandre , Lawley Trevor D. 
Published: February 2023

Abstract
The gut microbiota has been identified as a leading cause of irreproducibility in mouse models, but current resources are insufficient to address this core challenge in immunology research. Furthermore, although mouse models are central tools for biomedical science, it is not known how the bacteria in the mouse gut – important determinants of immunological phenotypes – affect their ability to recapitulate human disease. To better characterise the mouse gut microbiota and facilitate its functional and taxonomic comparison to the human microbiota, we developed the Mouse Microbial Genome Collection (MMGC), the most comprehensive representation of the global laboratory mouse microbiome to date. The MMGC is a repository of 276 genomes from cultured isolates and 18,075 non-redundant, near-complete metagenome-assembled genomes (MAGs) reassembled from 1,960 mouse metagenomes.
Using the MMGC, we define species-level signatures of inter-institutional variation in the mouse gut microbiota and provide a roadmap to achieve more relevant and reproducible mouse models. In addition, we confirm that while only 2.65% of bacterial species are common to human and mouse gut microbiotas, over 80% of annotatable functions are shared between hosts. The MMGC further enables the identification of functionally equivalent taxa in the mouse and human gut microbiotas, which we illustrate by comparing the pathways for butyrate synthesis and drug metabolism as proof-of-concept examples. In conclusion, the MMGC facilitates unprecedented insights into the mouse gut microbiota and enhances the use of mouse models in immunology research by providing access to the conservation status and taxonomic locations of microbial functions of interest.
Automated Large-Scale T Cell Isolation in a New Closed Cell Separation System
Authors: Posarac Vesna , Buck Chris A. , Gellner Savannah D. , Williamson Mark E. , Egeler Oliver , Rahbar Mona , Dalton Bob , Eaves Allen C. , Louis Sharon A. , Kokaji Andy I. 
Published: February 2023

Abstract
Large-scale T cell isolation is commonly performed in a wide range of laboratory settings, including for cell therapy and drug discovery research, as well as in core facilities as part of cell manufacturing and banking. However, current methods can be a significant bottleneck in a lab’s workflow, often requiring a full day just for sample processing and cell isolation. Furthermore, many applications have stringent requirements for purity, sterility, and standardization of the cell isolation procedure. To address these needs, we have developed RoboSep™-C, an instrument for efficient and automated cell isolation in a closed system.
RoboSep™-C automates the established EasySep™ technology for immunomagnetic cell separation to enable isolation of untouched T cells, CD4+ T cells, or CD8+ T cells from leukapheresis samples. To set up the system, the user follows the on-screen prompts to install a sterile single-use tubing set and load the recommended medium, cell isolation reagents, and starting leukopak. The instrument then performs all necessary cell processing steps including sample washing, cell labeling, magnetic separation, and cell concentration in a 50-minute protocol. Starting with samples ranging from 2.5 to 20 billion nucleated cells, we obtained purities of 95.4 ± 4.1%, 95.7 ± 2.5%, and 89.3 ± 3.5% for T cell (n=16), CD4+ T cell (n=14), and CD8+ T cell (n=12) isolation, respectively (mean ± SD). The recoveries of the isolated T, CD4+ T, and CD8+ T cells were 64.2 ± 12.4%, 67.0 ± 8.2%, and 54.3 ± 13.5%, respectively.
Automated isolation of high-purity T cells with RoboSep™-C enables researchers to scale up their operations and can be easily integrated upstream of existing T cell expansion, genome editing, and cryopreservation protocols.