bluuebunny/crossref_metadata_2025 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

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

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

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

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

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 (>95% CD3+/CD8+, >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

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

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 >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

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

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

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

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 <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

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

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.
High Capacity Immunomagnetic Isolation of Apheresis Samples in an Automated Closed System
Authors: Hsu Faye , Sa Silin , Yu Liping
Published: February 2023

Cell therapy development, including Chimeric antigen receptor (CAR)-T cells, involves many steps and requires processing on average 3 billion white blood cells or more. Automated large scale immunomagnetic cell isolation with high efficiency and throughput in a closed system is desired to enrich T cells from apheresis products.

Current automated, high capacity methods are time consuming, high cost, low yield and can potentially influence the physiology of the target cells. We have developed an automated closed magnetic separation platform, MARS® Bar to address these problems. This platform uses three uniquely designed magnetic separation modules and each magnetic module can perform positive or negative selection of target cells in a fluidic chamber at high flow rate (1mL/min per module). On this platform we have developed cell isolation reagents and program with minimal cell preparation. In the pilot study, we isolated 116 million CD3+ T cells from 267million CD45+ cells in a single LSR chamber on a single MARS® module. The entire automated process took less than 15 minutes to complete and resulted in > 95% purity and > 96% recovery of CD3+ T cells. The isolated cells were then activated and expanded, with results showing high proliferation and the dominant population being central memory T cells. The unique MARS® Bar matrix-free design has unlimited capacity because of the automated release of positively captured cells and very low carry-over. This was validated by processing greater than 3 billion leukocytes at 20 million cells per mL within the three modules in parallel and intermittent release of captured cells for collection. The MARS® Bar can be easily integrated into standard T cell production workflow.
High-Throughput and High-Dimensional Single Cell Analysis of Antigen-Specific CD8+ T cells
Authors: Jiang Ning , Ma Ke-yue , Schonnesen Alexandra A. , He Chenfeng , Xia Amanda , Sun Eric , Chen Eunise , Sebastian Katherine , Balderas Robert , Kulkarni-Date Mrinalini
Published: February 2023

Although critical to T cell function, antigen specificity is often omitted in high-throughput multi-omics based T cell profiling due to technical challenges. We describe a high-dimensional, tetramer-associated T cell receptor sequencing (TetTCR-SeqHD) method to simultaneously profile TCR sequences, cognate antigen specificities, targeted gene-expression, and surface-protein expression from tens of thousands of single cells. Using polyclonal CD8+ T cells with known antigen specificity and TCR sequences, we demonstrated over 98% precision for detecting the correct antigen specificity. We also evaluated gene-expression and phenotypic differences among antigen-specific CD8+ T cells and characterized phenotype signatures of influenza- and EBV-specific CD8+ T cells that are unique to their pathogen targets. Moreover, with the high-throughput capacity of profiling hundreds of antigens simultaneously, we applied TetTCR-SeqHD to identify antigens that preferentially enrich cognate CD8+ T cells in type 1 diabetes patients compared to healthy controls, and discovered a TCR that cross reacts between diabetic and microbiome antigens. TetTCR-SeqHD is a powerful approach for profiling T cell responses.
An Accessible Automatic Cell Counting Methodology Using Trainable Weka Segmentation in ImageJ Validated on Murine Microglia
Authors: Kataras Theo J , Koury Jeffrey , Singh Hina , Kaul Marcus
Published: February 2023

Counting cells is a cornerstone of tracking disease progression in neuroscience. A common approach for this process is having trained researchers individually select and count cells within an image, which is not only difficult to standardize but also very time-consuming. We present an automatic cell counting methodology which leverages the Trainable WEKA Segmentation (TWS) plugin available in the FIJI distribution of ImageJ (Schindelin 2012, Schneider 2012). TWS provides access to machine learning tools for object segmentation with an accessible graphical user interface. However, to analyze whole datasets requires further automation through the use of macros which we constructed within ImageJ maintain this high level of accessibility.

We compared our automatic counts to a published, hand counted dataset of Immunofluorescence stained mouse cortex microglia (Singh et al. 2020). For this analysis we used a wild-type (WT) control and a transgenic mouse model for HIV-induced brain injury (HIVgp120). HIVgp120 mice showed behavioral deficits, and increase in microglia numbers presumably caused the gp120 protein. 10X images were collected in the sagittal plane from brain slices of three 11–14 month-old male mice for each genotype.

The auto counting strategy with TWS replicated the increase in mean microglial density in images from the HIVgp120 mice (Manual p =0.0001; Auto p=0.0002). The auto count also correlates significantly with the manual counts in both genotypes (WT p= 2.297 e−07, adj. R2= 0.65; HIVgp120 p=2.591 e−04 adj. R2=0.423). The auto counting strategy took less than a fifth of the time for the expert manual count, allowing for the efficient exploration of large sets of image data.
Lumit&[trade] Immunoassays for Simple and Rapid Determination of Cytokine Release from Cell Culture Models
Authors: Lazar Dan F , Hsiao Kevin , Sondgeroth Casey , Gilden Julia , Kupcho Kevin , Thompson David , O’Brien Martha , Cali James
Published: February 2023

Rapid (< 70 min), add-and-read assays for cytokine determination were developed with Lumit™ immunoassay technology. Separate antibodies labeled with complementary SmBiT or LgBiT subunits bind to their shared cytokine target, resulting in proximity-driven reconstitution of NanoBiT luciferase and luminescence signal. Lumit immunoassays for human IL-1β, IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF-α exhibit LLODs < 10 pg/ml and linearity > 3 logs of analyte in 96-well format. Maximal S/B ratios are greater than 1000, and an assay signal T1/2 of 2 h accommodates batch plate processing. 384-well, assay performance (Z′ > 0.7) on treated human peripheral blood mononuclear cells (hPBMC) indicates the assay format is amenable to screening applications.

Assay reagents were added directly to cell wells for multiple culture models. For hPBMC, LPS, R848, or a combination of PMA and ionomycin (CSC) produced dose-and time-dependent release of cytokines with a wide range of responses, all measurable without sample dilution. In a mix of purified CD8+ T cells and target Raji B cells treated with the bispecific T-cell engager Blincyto®, IL-2, IFN-γ, and TNF-α release was observed with an EC50 of ~0.2 ng/ml and max S/B ratios of 82-, 168-, and 31-fold, respectively. For Th2 cells, significant release of IL-4 was observed in response to CSC treatment. An alternative to the no-transfer assay method, split-sample analysis, can be performed to easily determine the levels of multiple cytokines released in the same cell well.

The implementation of this novel detection chemistry will enable simple and rapid assay of cytokine release from cells for both low-and high-throughput applications, including quantitative assessments of T cell activation and differentiation.
StarBright Dyes: New Tools for Flow Cytometry
Authors: Green Angela M , Cichewiz Magdalena , Li Lei
Published: February 2023

StarBright Dyes are fluorescent nanoparticles, with superior brightness over conventional flow cytometry dyes. They have narrow excitation and emission peaks, hence low spillover, spreading and compensation, providing greater flexibility when building multiplex panels in flow cytometry. StarBright Dyes have been conjugated to numerous antibodies against surface markers found on human and mouse cells. High stability and batch consistency have been confirmed using the 5-laser and 27-detector ZE5 Cell Analyzer. The antibody conjugates also showed high reagent compatibilities, without a need for special cell staining buffers even in multiplex panels with additional conjugates and can be pre-mixed and used at a later date. Furthermore, they are able to reliably identify positive cells regardless of the staining buffer and conditions used, providing flexibility and helping researchers avoid false negatives. StarBright Dyes are new valuable tools to fulfill unmet needs in immunology-based flow cytometry.
Flt3 ligand expressed on the surface of T-cells contributes to effector function
Authors: Molina Megan Stanley , Stokes Jessica , Hoffman Emely , Simpson Richard J , Katsanis Emmanuel
Published: February 2023

Flt3 ligand (FL) is a growth factor that contributes to the homeostasis of the dendritic cell (DC) compartment. FL provides signals for differentiation, development, and survival by binding to the Flt3 receptor tyrosine kinase. Flt3 expression is maintained on DCs through their terminal differentiation. Endogenously, FL is produced by stromal cells and select immune cells, including T-cells and some DC subsets. FL may be expressed as a membrane-bound form (mFL), with no intracellular signaling domain, and may undergo cell-autonomous shedding to generate soluble FL. This is believed to contribute to a localized source of FL for those DC subsets, however T-cells do not express Flt3 and have no FL requirement. As such, it remains unclear why T-cells express mFL on their surface. Interestingly, FL-driven DCs are more steady state-like, secreting less pro-inflammatory cytokines, and can suppress graft-versus-host disease (GvHD). We hypothesized that mFL on T-cells may regulate effector responses through binding to Flt3 on DCs. To test this, we compared GvHD in a murine bone marrow transplant model using WT compared to FL−/− donor cells. Engraftment, T-cell reconstitution, and activation were monitored weekly. We also performed mixed leukocyte reactions (MLR) to measure T-cell proliferation in WT versus FL−/− T-cells. Infusion of FL−/− donor cells resulted in significantly worse GvHD and higher expression of T-cell activation markers CD25 and CD69 in vivo. The absence of mFL also resulted in significantly enhanced alloreactive T-cell proliferation in vitro. These studies indicate that mFL expressed on the surface of T-cells can alter their effector function, which may be a result of the engagement of mFL with Flt3 receptor expressed on DCs.
Vγ2+ gamma-delta (γδ) T cells promotes the survival of skin allografts in the presence of anti-CD40L induced transplantation tolerance
Authors: Lal Girdhari , Giri Shilpi
Published: February 2023

Donor-specific transfusion (DST) and anti-CD40L treatment is one of the best tolerogenic strategies for promoting the survival of allografts. The effect of DST and anti-CD40L treatment on the phenotype and function of most of the innate and adaptive immune cells are known. Gamma delta (γδ) T cells uniquely possess features of both innate and adaptive immune cells and perform crucial functions during cancer and autoimmune diseases. Recent reports have suggested that γδ T cells promote skin allograft rejection but their contribution during costimulatory blockade induced skin allograft survival is not known. In the present study, we show that depletion of γδ T cells or specifically Vδ2+ γδ T cells led to acute rejection of skin allografts even with DST and anti-CD40L mAb treatment. Tolerogen treatment increased the expression of regulatory receptor CD39 on Vδ2+ γδ T cells. Tolerogen treatment reduced IFN-γ expression in Vγ2+ γδ T cells which in-turn suppressed the Th1 differentiation of naive CD4+T cells. Depletion of Vγ2+ γδ T cells induced hyper IFN-γ response in tolerogen-treated allograft recipients. Tolerogen-treated and Vγ2+ γδ T cells-depleted recipients showed enhanced infiltration of activated macrophages in the skin allografts. Adoptive transfer of Vγ2+ γδ T cells purified from tolerized mice in naive mice promoted the survival of skin allografts. Taken together, our results showed that Vγ2+ γδ subset of T cells promote costimulatory blockade-induced prolonged survival of allografts and suggest its importance in adoptive cellular therapy to control the allograft rejection.
The effects of UW Solutions in preventing ischemia induced apoptosis of retinal ganglion cells after whole eye transplantation
Authors: Khatter Neil J. , Matlock Tyler , Owens Charles R. , Li Bing , Wong Yong , Huang Christene A. , Nickells Robert W. , Su An-Jey A. , Washington Kia M.
Published: February 2023

Background

Retinal ganglion cell (RGC) death from ischemia reperfusion injury and optic nerve transection are challenges to successful restoration of vision by whole eye transplantation (WET). We examined whether University of Wisconsin Solution (UW) or a modified version which includes valproic acid and BaCl2 (mUW) can preserve RGCs in a WET model.

Methods

24 hemifacial rat flaps prepared for WET were flushed with heparinized saline, UW, or mUW. After 85 minutes of ischemia, donor eyes were injected with the same solutions and prepared for immunohistochemistry (IHC) analysis before (n=6/group) or POD7 after (n=3/group) syngeneic WET. Donor and untreated non-ischemic naïve (n=4) retinas were stained for Brn3a and p-cJun (PNJ) to assess RGC viability and apoptosis respectively.

Results

After 85 minutes of ischemia, all donor eyes across treatments (saline, UW, or mUW), had equal levels of Brn3a+ and PNJ+ expression (P>0.05). Naïve eyes averaged ~100k Brn3a+ and ~18k PNJ+ RGCs. At POD7, saline and UW donor eyes each had ~20% loss in Brn3a+ RGCs as compared to control naïve eyes (P<0.05). At POD7, apoptotic signaling increased in both UW and saline donor eye, doubling in PNJ+ signal compared to non-ischemic naïve eyes (P<0.05).

Conclusions

Using a novel system to asses RGC degradation and apoptosis in an experimental rodent WET model, we found that UW and mUW solutions as formulated did not preserve RGCs after WET, despite reports of their anti-ischemic properties in other studies.
Durvalumab and Lenvatinib in Participants With Locally Advanced and Metastatic Hepatocellular Carcinoma (Dulect2020-1)
Authors: Feng Hao , Tong Huan , Xia Qiang
Published: February 2023

Background:

This study will evaluate the safety and efficacy of durvalumab in combination with lenvatinib in participants with locally advanced hepatocellular carcinoma before liver transplant and metastatic unresectable HCC. The primary hypothesis of this study is that patients with locally advanced HCC could benefit from durvalumab plus lenvatinib before liver transplant; patients with metastatic unresectable HCC could also benefit from durvalumab plus lenvatinib concerning: 1) Progression-Free Survival (PFS); or recurrence-free survival (RFS) if patients with locally advanced HCC underwent liver transplant; 2) Objective Response Rate (ORR), and 3) Overall survival (OS).

Methods:

The investigators design a clinical study to explore whether the combination above as a treatment in patients with advanced and recurrent endometrial carcinoma could prolong PFS and analyze potential immune biomarkers of therapeutic response. This trial is designed as a prospective, open-label study for patients with locally advanced hepatocellular carcinoma before liver transplant and metastatic unresectable HCC (NCT04443322).

Results:

20 patients with pathologically or cytologically proved locally advanced or metastatic hepatocellular carcinoma were recruited. The combination therapy of Lenvatinib 80–120mg daily orally and durvalumab 1500mg by IV infusion every 4 weeks were performed. PFS and RFS were evaluated as the primary endpoint. ORR, OS, and Percentage of Participants who Experience an Adverse Event (AE) were assessed as the secondary endpoints.

Conclusion:

Durvalumab, combined with Lenvatinib in participants with locally advanced and metastatic hepatocellular carcinoma, is safe and effective.
Distinct phenotype of antibody-suppressor CXCR5+CD8+ T cells
Authors: Zimmerer Jason M , Han Jing , Hart Madison , Chaudhari Sachi , Peterson Chelsea M , Bumgardner Ginny L
Published: February 2023

The novel CXCR5+CD8+ T cell subset has been shown to inhibit alloantibody production (termed CD8+ TAb-supp cells) but do not reject allografts. In contrast, CXCR3+CD8+ T cells reject allogeneic transplants (but do not inhibit antibody). Recent literature indicates there are many CXCR5+CD8+ T cell subsets with different effector functions (anti-viral, anti-tumor, anti-autoimmune, or antibody enhancement). By investigating CD8+ TAb-supp cell by RNA-seq and flow cytometry, we aimed to determine a profile that distinguishes this novel subset. To investigate this, C57BL/6 (H-2b) were transplanted with FVB/N (H-2q) hepatocytes and CD8+ T cell subsets were analyzed on day 7. Alloprimed CXCR5+CD8+ T cells were compared to naïve CD8+ T cells and alloprimed CXCR3+CD8+ T cells. Fluorescence minus one controls were used to determine background staining (flow cytometry). RNA-seq analysis show that 1670 transcripts were upregulated or downregulated when comparing between flow-sorted alloprimed (CD62L−CD44+) CXCR5+CD8+ T cells or naïve (CD62L+CD44−) CD8+ T cells. Transcripts of note include Bcl-6 (3.3-fold upregulated), CXCR3 (8.4-fold downregulated), and S1PR3 (140-fold upregulated). Flow cytometry analysis suggests that CD8+ TAb-supp cells are short-lived effectors cells based on the expression of CD44, KLRG1, and CD122 (not CD62L or CD127). An expansive flow analysis panel comparing differences in transcript and protein expression between alloprimed CXCR5+CD8+ TAb-supp cells and other reported CXCR5+CD8+ T cell subsets indicates that the absence of PD-1, IL-10, and FoxP3 expression distinguishes CD8+ TAb-supp cells from all reported CXCR5+CD8+ T cell subsets (including CD8+ T follicular regulatory cells).
MHC II expression by donor bone marrow-derived macrophages underpins neuroinflammation and cognitive deficits during murine chronic GVHD
Authors: Adams Rachael C , Shaik Samreen N , Llanes Genesis T , Carter-Cusack Dylan , Blazar Bruce B , Vukovic Jana , MacDonald Kelli PA
Published: February 2023

Chronic graft-versus-host disease (cGVHD) remains the leading cause of non-relapse mortality after allogeneic stem cell transplantation (SCT) for haematological malignancies. Manifestations of GVHD in the central nervous system (CNS) present as neurocognitive dysfunction in a significant proportion of patients. Neuroinflammation in the cortex of mice and humans in acute GVHD is dependent on host resident microglia-derived TNF. However, the mechanisms driving cGVHD in the CNS are yet to be elucidated. Here, our studies of murine cGVHD revealed a hippocampal-dependent spatial learning and memory deficit associated with inflammation and persistent IFN-γ upregulation. Hippocampal RNA sequencing identified perturbations to structural and functional synapse-related gene expression, in the context of upregulated genes associated with antigen presentation and IFN-γ responses. Brain T cell infiltration was initially dominated by CD8 T cells with an eventual expansion in CD4 T cells, in parallel with a marked infiltration of MHC Class II-expressing bone marrow-derived macrophages (BMDM). After day 70, BMDM constituted 50% of the brain macrophage pool and exhibited a transcriptional signature distinct from host resident microglia. Transfer of MHC class II–deficient BM grafts resulted in attenuated neuroinflammation, implicating donor BMDM as critical mediators of CNS cGVHD. Our identification of the hippocampus as a cGVHD target, and disease mediators distinct from those in the acute phase, highlights the necessity to pursue alternative therapeutic targets for late-stage neurological manifestations post-SCT.
Intranasal adjuvant immunotherapy increases survival in murine metastatic Lymphangioleiomyomatosis (LAM)
Authors: Amosu Mayowa , Maisel Katharina
Published: February 2023

Pulmonary lymphangioleiomyomatosis (LAM) is a slow progressing, metastasizing neoplasm primarily affecting women of reproductive age. LAM is characterized by the abnormal growth of smooth muscle-like cells leading to cystic destruction of the lungs. Rapamycin, the only FDA approved treatment for LAM, slows disease progression but patient responses are incomplete. Therefore there is a critical need for new LAM treatments. Research has shown that LAM has several characteristics of cancer including suspected immune suppression. We and others recently found that LAM may be treated using immune checkpoint inhibitors (ICI), a common cancer immunotherapy. Here, we investigate immune-activating adjuvants as local anti-LAM immunotherapies.

Using an established murine model of metastatic LAM, we determined survival after twice weekly intranasal treatments of CpG, PolyI:C, or MPLA. We used flow cytometry and immunofluorescence to determine how adjuvants alter the immune response in LAM.

We found that intranasal treatment with CpG and PolyI:C, but not MPLA, increased survival in mice with LAM. In more progressive LAM disease, we observed an increase in activated CD8 T cells, and a decrease in B cell, macrophage, and eosinophil infiltration in lungs of CpG treated vs untreated LAM mice. We also observed increased expression of MHC-II on alveolar macrophages but decreased numbers of migratory dendritic cells in CpG treated LAM lungs.

These results suggest that CpG immunotherapy in LAM lungs activates cytotoxic lymphocytes while tempering other inflammatory cells like eosinophils. Our findings also suggest that LAM lungs may respond favorably to adjuvant immunotherapy, and that combination ICI-adjuvant therapy could be synergistic.
Humanized mice challenged with TNBC tumor develop graft versus host disease and anemia limiting vaccine immunotherapy studies
Authors: Bommireddy Ramireddy , Munoz Luis E , Pack Chrstopher , Ramachandiran Sampath , Reddy Shaker , Selvaraj Periasamy
Published: February 2023

Breast cancer is the leading cause of death among female cancer patients. Our lab has developed a therapeutic cancer vaccine approach for triple negative breast cancer (TNBC). In this study, we investigated whether vaccine developed from a human TNBC cell line could effectively induce antitumor immunity, which control the growth of tumors in humanized mice implanted with the human TNBC cell line. Human CD34+ HSC implanted NSG-SGM3 from JAX labs and NOG-EXL mice from Taconic Biosciences used for the studies. Humanized NSG-SGM3 mice were vaccinated with 100 mg of the vaccine once every 2 weeks (3 doses). Control mice were injected with vehicle (PBS). One week after the last vaccination, mice were challenged with MDA-MB-231 TNBC cells subcutaneously in the hind flank. Body weight and tumor growth were monitored. All the mice in both groups have developed tumors, splenomegaly and metastatic lesions in the liver, lungs and spleen. However, all the mice also developed GvHD and need to be euthanized before completion of the study. We have also observed an allo-response (CD69+ T cells in control mice with MDA-MB-231 tumors) to the HLA-matched tumor cells but the tumors eventually developed into metastatic disease affecting multiple organs (liver, lungs and spleen) suggesting that the allo-response is not effective against the tumor. Since the tumor-bearing mice developed GvHD and becoming anemic even in a specific pathogen-free barrier facility, future studies will need to be tested in humanized mice which do not develop GvHD and live longer for carrying out vaccine immunotherapy studies. Funding: NIH/NCI R01 CA202763 (PS & CP).