bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulatory T cells tonically inhibit spontaneous activation of naturally arising memory-phenotype CD4+ T lymphocytes
Authors: Li Jing , Yang Ziying , Kawajiri Akihisa , Sato Kosuke , Tayama Shunichi , Ishii Naoto , Kawabe Takeshi 
Published: November 2024

Abstract
Foxp3- CD4+ T lymphocytes consist of naïve (CD44lo), foreign antigen-specific memory, and self antigen-driven memory-phenotype (MP; both CD44hi) cells at homeostasis. We recently showed that self-reactive MP cells spontaneously proliferate and differentiate into the T-bet+ subset. How excess proliferation and Th1 differentiation of MP cells are inhibited in steady state remains unclear. Regulatory T cells (Tregs) can inhibit several immune responses, posing a possibility that the same lymphocytes may suppress spontaneous MP cell activation. Here we show using Foxp3-diphtheria toxin receptor-transgenic mice that both preexisting MP and naïve CD4+ T cells spontaneously proliferate and differentiate into Th1 cells upon Treg depletion. MP as compared to naïve CD4+ T cells more rapidly proliferate and differentiate three to five days post Treg depletion whereas these responses are mainly attributed to by the latter T cells at a later time point. Moreover, we demonstrate that these early MP proliferation and differentiation are driven in a manner dependent on CD28 and IL-2 signals, respectively. Furthermore, our data suggest that such activation of MP and naïve CD4+ T cells contribute to development of multi-organ inflammation at early and later time points, respectively. Together our results argue that Tregs tonically inhibit early, spontaneous proliferation and Th1 differentiation of MP CD4+ T lymphocytes, thereby contributing to maintenance of T cell homeostasis.
You're (Cyto)Toxic: HDAC5 controls the activation of cytotoxic CD8+ intraepithelial lymphocytes
Authors: Borregard Megan , Jabri Bana , Ciszewski Cezary 
Published: November 2024

Abstract
In Celiac Disease, upregulation of IL-15 by distressed intestinal epithelial cells results in the pathogenic reprogramming of cytotoxic CD8+ intraepithelial lymphocytes (IE-CTLs), which then promote tissue destruction. We found that IL-15 induced major transcriptional changes associated with effector function in IE-CTLs, but did so without impacting chromatin accessibility. Here, we explore how the intestinal microenvironment controls IL-15-mediated activation of IE-CTLs without restructuring their epigenetic landscape. We found that butyrate, a known immunomodulatory molecule, reduced expression of Granzyme B and NKG2D in ex vivo human IE-CTLs. Further, butyrate broadly impacted the expression of genes linked to survival and activation in a histone deacetylase (HDAC)-dependent manner. Using small interfering RNA, we determined that HDAC5 controlled the IL-15-mediated activation of IE-CTLs without impacting their survival. HDAC5 has little to no deacetylase activity against histone tails, and our data suggest that it remains localized in the cytoplasm following stimulation. We believe that HDAC5 may deacetylate key signaling molecules to promote their activation, and thus regulate the IL-15-mediated licensing of IE-CTLs. Understanding how HDAC5 controls activation of IE-CTLs could shed light on novel therapeutic options that specifically target IE-CTL activation responses in inflammatory T cell disorders, like Celiac Disease, without impacting their survival in the tissue.
CARD11-dependent signaling in T cells is critical for the activation of multiple AP-1 transcription factor complexes.
Authors: Bauman Bradly , Stinson Jeffrey , Frank Andrew , Sukumar Gauthaman , Dalgard Clifton , Snow Andrew 
Published: November 2024

Abstract
The CARD11 scaffold enables TCR-induced NF-κB, mTORC1 and JNK signaling. While the requirement of CARD11 for NF-κB stimulation is well known, it remains unclear if/how CARD11 influences other transcription factors (TFs) essential for T cell activation: AP-1 and NFAT. We sought to delineate the importance of CARD11 in activating AP-1- or NFATc1-dependent transcription in human T cells, employing mitogen-activated wild-type (WT) or CARD11 KO Jurkat T cells. RNA-seq analysis revealed significant downregulation of AP-1-mediated transcriptional programs in CARD11 KO cells, with a concomitant increase in NFATc1 expression. Although JNK inhibition in WT cells had a surprisingly minimal impact on AP-1 activation, loss of CARD11 substantially reduced mitogen-induced AP-1 component expression and activity, encompassing all canonical JUN and FOS family members. We further demonstrated that deleterious CARD11 variants derived from CARD11-associated Atopy with Dominant Interference of NF-κB Signaling (CADINS) disease patients also impaired AP-1 function. Conversely, JNK inhibition in WT T cells enhanced NFATc1 activity with concomitant upregulation of the NFAT-dependent TH2 master regulator GATA3, congruent with enhanced GATA3 induction in CADINS patient T cells. Our novel findings indicate that CARD11 governs the activation of AP-1 and NFAT TF complexes originally thought to be induced via CARD11-independent pathways, with implications for human T cell differentiation and function.
Resolving haplotype variation and complex genetic architecture in the human immunoglobulin kappa and lambda chain loci in individuals of diverse ancestry
Authors: Engelbrecht Eric , Rodriguez Oscar , Shields Kaitlyn , Schultze Steven , Tieri David , Jana Uddalok , Lees William , Smith Melissa , Watson Corey 
Published: November 2024

Abstract
Immunoglobulins (IGs), critical components of the human immune system, are composed of heavy and light protein chains encoded at three genomic loci. The IG Kappa (IGK) chain locus consists of two large, inverted segmental duplications. The complexity of IG loci has hindered effective use of standard high-throughput methods for characterizing genetic variation within these regions. To overcome these limitations, we leverage long-read sequencing to create haplotype-resolved IGK assemblies in an ancestrally diverse cohort (n=36), representing the first comprehensive description of IGK haplotype variation. We identify extensive locus polymorphism, including novel single nucleotide variants (SNVs) and a common novel ∼24.7 Kbp structural variant harboring a functional IGKV gene. Among 47 functional IGKV genes, we identify 141 alleles, 64 (45.4%) of which were not previously curated. We report inter-population differences in allele frequencies for 14 IGKV genes, including alleles unique to specific populations within this dataset. In addition, we identify haplotypes carrying signatures of gene conversion that associate with enrichment of SNVs in the IGK distal region. Finally, we identify extensive polymorphism in the IG Lambda (IGL) locus, including copy number variation for genes encoding IGLC and IGLJ genes. These data lay a foundation for advancing our understanding of how genetic variation in IG loci impacts expressed antibody repertoires in human populations.
Diversity and affinity from plasma vs. B cells for monoclonal antibody development
Authors: Hawksworth David , Moore Jeff , Larkin Isaac 
Published: November 2024

Abstract
Diagnostic assay development requires a diverse panel of high affinity antibodies. Historically, our lab has used splenic CD19+ B-cells to generate mouse hybridoma cell lines. However, literature suggests that, as a group, terminally differentiated, CD138+ plasma cells secrete the highest affinity antibodies, making these cells an attractive target for hybridoma development. To compare the use of these two lymphocyte populations for generating mouse hybridomas, three fusion experiments were completed whereby cells were separately isolated and fused. Hybridomas were plated in semi-solid media, expanded, and selected based on size and antibody secretion. An average of 246 (+ 73) plasma cell hybridoma colonies were available for selection, per 1x106 specific lymphocytes used for fusion, compared to 19 (+ 13) B cell hybridoma colonies. Cells from selected colonies were transferred to 96-well plates, grown for 7 days and the antibodies screened for antigen binding by enzyme immunoassay. Across the three fusion experiments, 40 (+19)% of the wells with growth from the plasma cell hybridomas were antigen reactive, vs. 6 (+ 0.5)% from the B cell hybridomas. Our data suggests a higher fusion efficiency from the plasma cell population, which allows for a greater sampling of the mouse immune repertoire, leading to identification of more antigen specific antibodies with a wider range of affinities and greater sequence diversity.
Immune dynamics in response to risk group 4 pathogens in nonhuman primate models
Authors: Rivera Deja , Barr Bobbi , Marketon Anthony , Bernbaum-Culter Rebecca , Pahar Bapi , Anthony Scott 
Published: November 2024

Abstract
Despite the critical importance of emerging infectious disease research, the underlying immune responses to risk group 4 pathogens remain largely understudied relative to many other pathogens. Spectral flow cytometry has dramatically increased the capacity of fluorescence-based cell analyses, allowing for the development of comprehensive panels across a variety of cell types. We have developed a 33-color phenotypic panel that incorporates a plethora of markers to simultaneously identify numerous distinct cellular populations within circulation and in tissue with a deliberate and extensive characterization of monocytes, dendritic cells, and tissue-resident T cells. This panel utilizes multiple approaches to accurately identify appropriate cell markers that can be easily distinguished with minimal to no compensation, despite containing fluorochromes with similar excitation and emission spectra. Importantly, this high-complexity panel was implemented on both fresh and frozen samples of whole blood, peripheral blood mononuclear cells (PBMCs), and cells isolated from multiple peripheral tissues across several nonhuman primate (NHP) models and on frozen human PBMCs, making this a broad cross-reactive panel enabling direct comparisons among NHP species and with humans. This new phenotypic flow panel allows for broad insight into underlying immune responses to infections across species while remaining amenable to additional potential markers of interest.
StarBright Dyes — Expanding the Availability and Choice of Fluorophores to Improve Immunophenotyping Data in Veterinary Studies
Authors: Sanderson Sharon , Rosenwasser Oliver , Blundell Michael , Long Tracey 
Published: November 2024

Abstract
Flow cytometry is an important application for veterinary studies that is commonly used for diagnostics, drug screening, and for the fundamental understanding of veterinary diseases. However, the range of fluorophores that flow cytometry veterinary antibodies are conjugated to is limited. This restricts the choice available for multiplexing panels and therefore panel size, which is especially important for rare samples as the information that can be determined from a single sample will be reduced. In addition, less choice means that best practice for panel design cannot always be followed, often resulting in poor cell resolution and data quality.
To address this problem, Bio-Rad is expanding the range of antibodies that are conjugated to StarBrightTM Dyes, to include canine, bovine, and porcine targets. Antibodies conjugated to StarBright Dyes are bright, easy to use, and will work with most protocols and cytometers. This means you have the choice and flexibility to follow best practice and make bigger panels, getting better results, saving time and resources.
We present data showing multiplexing panels on canine, bovine, and porcine peripheral blood, acquired on the ZE5 Cell Analyzer from Bio-Rad. These immunophenotyping panels include antibodies which are conjugated to traditional fluorophores as well as new StarBright Dyes. We demonstrate how multiple cell populations and subpopulations can easily be detected, such as B cells, T cells, and natural killer cells.
Enhanced Annotation of CD45RA to Distinguish T cell Subsets in Single Cell RNA-seq via Machine Learning
Authors: Ran Ran , Brubaker Douglas 
Published: November 2024

Abstract
T cell heterogeneity presents a challenge for accurate cell identification, understanding their inherent plasticity, and characterizing their critical role in adaptive immunity. With the advent of single-cell RNA sequencing (scRNA-seq), researchers can now investigate the gene expression profiles of these surface proteins at the single-cell level, having a deeper understanding of cell identity. However, CD45RA, the isoform of CD45 which distinguish between naive/central memory T cells and effector memory/effector memory cells re-expressing CD45RA T cells, cannot be well profiled by scRNA-seq due to the difficulty in mapping short reads to genes. In order to facilitate cell type annotation in T cell scRNA-seq analysis, we employed machine learning and trained a CD45RA+/- classifier on single-cell mRNA count data annotated with known CD45RA antibody levels provided by cellular indexing of transcriptomes and epitopes sequencing (CITE-seq) data. Among all algorithms we tested, the trained support vector machine (SVM) with a radial basis function (RBF) kernel with optimized hyperparameters achieved a 99.96% accuracy on an unseen dataset. The multilayer Perceptron (MLP) classifier, the second most predictive method overall, also achieved a decent accuracy of 99.74%. Our simple yet robust machine learning approach provides a valid inference on the CD45RA level, assisting the cell identity annotation and further exploring the heterogeneity within human T cells.
Unleashing the Power of γδ-T Cells with REAlease Technology
Authors: Brühl Vanessa , Donner Christin , Dhana Ermanila , Räty Lotta , Hildalgo Jose , Höher-Peters Susanne , Winkels Gregor 
Published: November 2024

Abstract
γδ-T cells represent a unique subset of immune cells that share characteristics of the innate and adaptive immune system. They are not MHC-restricted, making them promising candidates for allogeneic CAR T cell therapies. Enrichment of γδ T cells allows to simplify research, improve understanding of their biology and facilitate developments for therapeutic applications. The cutting-edge REAlease technology offers a direct means of enriching unlabeled cells from diverse blood materials. The option of full automation contributes to the standardization of routine separations, concurrently minimizing hands-on time. Evaluation of separation performance and functionality was conducted through flow cytometric analyses and in-vitro assays. Demonstrating the utility of the newly developed REAlease γδ T-cell MicroBead Kits, we achieved high-purity enrichment from PBMC, BC, WB and leukopak. The ratio of γδ T subtypes in enriched fractions was analyzed,, as well as the activation status. Isolated cells showed no expression of activation marker CD69 and remained inactive in culture. Stimulation effectively activated the cells showcasing their full functionality. Subsequent analysis examined cytokine secretion in non-stimulated and stimulated cells. The isolation strategy presented minimizes cell manipulation and reduces interference with downstream processes. It can thus make an important contribution to the progress of disease research and the exploration of new therapeutic possibilities.
An Alternative Immune Cell Labeling System Based on the New Two-Dimensional Nanomaterials MXenes
Authors: Giro Linda , Fusco Laura , Gazzi Arianna , Benjamin Chacon , Shuck Christopher , Ley Klaus , Orecchioni Marco , Gogotsi Yury , Delogu Lucia 
Published: November 2024

Abstract
Deepening our understanding of immune cell behavior is vital for creating safe, effective treatments, particularly as immune cell therapy, also known as cellular immunotherapy or cell-based immunotherapy, represents a groundbreaking approach in the treatment of various diseases, including cancer. Transition metal carbides and nitrides (MXenes), a novel family of 2D nanomaterials, show promise as advanced trackers for immune cells—key for precise diagnostics and therapies. Traditional cell labeling methods have stagnated due to limited chemical options, impeding progress in applied medicine. Moreover, these methods are incompatible with single-cell mass cytometry by time-of-flight (CyTOF), a globally adopted technology that improves classical flow cytometry. We propose an innovative solution utilizing MXenes to overcome these challenges. Our method, Label-free sINgle-cell tracKing of 2D matErials by mass cytometry (LINKED), leverages a novel, biocompatible, multiplexed, label-free detection approach via CyTOF and Mass Ion Beam Imaging by Time-of-Flight (MIBI-TOF). This technique overcomes chemical limitations and integrates seamlessly with CyTOF, allowing for nanomaterial detection and simultaneous measurement of diverse immune cell and tissue features. Our work promises to advance immunological research significantly, offering refined cell labeling and tracking techniques crucial for the advancement of translational medicine.
Single cell analysis of melanoma reveals a prognostic myeloid cell signature associated with survival and response to immunotherapy in advanced melanoma
Authors: Ahmad M Usman , Heo Lyong , Nair Ramesh , Sharma Saurabh , Kirane Amanda 
Published: November 2024

Abstract
INTRODUCTION: We sought to elucidate myeloid sub-type differences to characterize biology and support therapeutic strategies in melanoma patients with diverse biogeophaphies.
METHODS: scRNA-seq of melanoma were combined from 6 datasets with 126 patients. After integration, cells were clustered with scCCESS, using an artificial neural network. We conducted Recursive Partitioning Analysis (RPA), differential expressed gene (DEG) analysis, and pseudobulk comparison. We validated the signature using MuSiC deconvolution on bulk RNA data.
RESULTS: 102,971 single cells from 126 patients generated myeloid cells of 6 cell types. RPA analysis of 42 patients with melanoma brain metastases revealed a single myeloid population associated with median survival months (13 vs 32 vs 52, p=0.029). Partial Response (p=0.010) and adjuvant targeted therapy (p=0.036) differed. Myeloid cell type 1 was enriched with CLEC10A, AXL, and MHC Class II genes. 42 patients on nivolumab were used to validate signature with on treatment biopsy with improved median survival months (13.25 vs 30, p=0.046) and Partial Response (p&lt;0.001). 19 patients shifted high infiltration groups during treatment with 11% dropping, 44% increasing, and 47% remaining high.
CONCLUSIONS: Increased infiltration of myeloid cells showed improved survival and treatment response in melanoma brain metastases. Validation of the signature showed an association with nivolumab response and survival.
Efficacy of IFN-g, CD40L, and poly(I:C) treated bone marrow-derived macrophages in murine mammary carcinoma
Authors: Kurt Robert , Roberts Meghan , Finn Joshua , Lass Melissa , Oviedo-Bermudez Ernesto 
Published: November 2024

Abstract
Here we explored methods to generate anti-tumor bone marrow-derived macrophages (BMDM) and how delivery of the BMDM at early tumor sites would impact disease progression. BMDM treated with IFN-g, CD40L, poly(I:C), and a combination of the three were assessed. Treatment with CD40L alone had no significant impact on cytokine production, phagocytic activity, or MHC expression by the BMDM. Treating BMDM with IFN-g impacted IL-1b expression as well as MHC Class II and CD80 expression. While poly(I:C) alone had a greater impact on the BMDM than IFN-g alone, treatment with poly(I:C), IFN-g, and CD40L had the greatest impact on the BMDM. Treatment with all three agonists resulted in increased IL-1b, TNF-a, and IL-12 expression, decreased expression of arginase and mrc, increased phagocytic activity, increased nitrite production, increased MHC Class II and CD80 expression, and these BMDM significantly impacted growth of two murine mammary carcinoma models; EMT6 and 168. However, the BMDM had no impact on progression of the 4T1 mammary carcinoma. Interestingly, BMDM treated with poly (I:C) alone significantly decreased growth of the EMT6 tumor, had no impact on growth of the 168 tumor, and enhanced growth and metastasis of the 4T1 tumor. Collectively, these data show that treating the BMDM with poly(I:C) alone exhibited varying results depending upon the tumor type, and that treating BMDM with poly(I:C), IFN-g, and CD40L generates BMDM with more consistent anti-tumor activity.
Target specificity in the ADAR editing landscape explains the unique immunoregulatory function of the interferon-inducible isoform ADAR1-p150
Authors: Pfaller Christian , Herrmann Maike , Krebs Yvonne 
Published: November 2024

Abstract
The adenosine deaminase acting on RNA (ADAR) family includes three enzymes, ADAR1-p150, ADAR1-p110, and ADAR2 that bind endogenous and pathogen-derived double-stranded RNA (dsRNA) and convert adenosine (A) to inosine (I). This process called A-to-I editing disrupts dsRNA structures and counteracts activation of innate immune sensors MDA-5 and PKR, preventing autoimmune disorders like Aicardi-Goutieres-Syndrome. We and others have shown that the interferon-inducible ADAR1-p150, but not the other ADARs, protects cells and organisms from aberrant innate immune activation by self-derived dsRNA, even though A-to-I editing is performed by all three ADARs. We hypothesized that ADAR1-p150 edits specific transcripts that cannot be edited by the other enzymes. Here we present a novel approach to quantify RNA editing in transcriptomic data. Our tool TSniffer detected more than 40,000 regions of A-to-I editing distributed over the entire human transcriptome, affecting 30% of protein-coding transcripts and 10% of long non-coding RNAs. Differential editing frequencies in cell lines with knock-outs for the different ADARs revealed transcripts exclusively edited by ADAR1-p150. In contrast, editing by ADAR1-p110 and ADAR2 was cooperative and more redundant. Our analyses fill gaps of knowledge about the A-to-I editing landscape, highlight the dominant immunoregulatory function of ADAR1-p150, and provide new insights in the role of aberrant A-to-I editing in disease development.
The FLRT3-UNC5B Pathway is a Novel Regulator of T Immunosurveillance
Authors: Flies Dallas , Yan Chuan , Yang Qiqi , Arbitman Steven , Fitzgerald Daniel , Sharee Sasan , Shaik Jahangheer , Bosiacki Jason , Myers Kayla , Paucarmayta Ana , Johnson Dorothy , O'Neill Tom , Cusumano Zachary , Langermann Solomon , Langenau David , Patel Shashank 
Published: November 2024

Abstract
Our understanding of molecular cell-surface interactions that control T cell responses in cancer remains limited despite many advances in the field. We hypothesized that tumor cells take advantage of developmental protein-protein interactions such as axon guidance interactions that could inhibit or repel tumor-immune interactions in the TME to evade T cell anti-tumor immunity. We used gain-of-function screening to evaluate a gene-set of axon guidance proteins for inhibitory function in human T cells and identified and validated Fibronectin and Leucine-Rich Transmembrane protein-3 (FLRT3) as an inhibitor of T cell activity. FLRT3 inhibited human T cell function and promoted tumor growth in humanized tumor cell-derived xenograft (CDX) models. FLRT3 inhibited T cells through interaction with Unc-5 Netrin Receptor B (UNC5B) expressed on activated T cells. A FLRT3 monoclonal antibody designed to specifically block FLRT3 interactions with UNC5 family proteins reduced tumor growth in CDX models that was dependent on T cells. FLRT3 blockade also enhanced CAR-T and BiTE + T cell activity and infiltration in humanized zebrafish tumor models. These studies support the notion that aberrant axon guidance protein expression in the TME act as T cell checkpoints, and the FLRT3:UNC5B pathway can be targeted therapeutically to improve T cell-based immunotherapy activity and infiltration.