bluuebunny/crossref_metadata_2025_split huggingface dataset:
Choline acetyltransferase-expressing T cells are required to control chronic viral infection
Authors: Cox Maureen Ann , Mak Tak 
Published: January 2023

Abstract
Although widely studied as a neurotransmitter, recent advances have revealed an important role for T-cell derived acetylcholine (ACh) in regulating immunity. However, the role of lymphocyte-derived ACh in viral infection is unknown. Here, we show that the enzyme choline acetyltransferase (ChAT), which catalyzes the rate-limiting step of ACh production, is robustly induced in both CD4+ and CD8+ T cells during lymphocytic choriomeningitis virus (LCMV) infection in an IL-21-dependent manner. Deletion of chat within the T-cell compartment ablated vasodilation in response to infection, impaired the migration of anti-viral T cells into infected tissues, and ultimately compromised the control of chronic LCMV-Cl13. Our results reveal a genetic proof-of-function for ChAT in T cells during viral infection and identify a pathway of T-cell migration that sustains anti-viral immunity.
IL-9high GATA3low ILC2s are potent inducers of allergic airway inflammation
Authors: Schmitt Pauline , Duval Anaïs , Roga Stéphane , Camus Mylène , Burlet-Schiltz Odile , Gonzalez-de-Peredo Anne , Cayrol Corinne , Girard Jean-Philippe 
Published: January 2023

Abstract
IL-9 is key driver of chronic and allergic inflammation at mucosal surfaces, with important roles in the activation and survival of mast cells and type 2 innate lymphoid cells (ILC2s). IL-9-reporter mice have established T cells (Th9) and ILC2s as major sources of IL-9 in vivo. The discovery that TGF-β and IL-4 cooperate for induction of Th9 cells has been an important breakthrough. However, the signals that induce IL-9high ILC2s remain incompletely characterized.
Here, we show that IL-33, a critical activator of ILC2s, with important roles in type-2 innate immunity and allergic inflammation (Cayrol, Duval et al., Nature Immunology 2018) synergizes with TNF-family cytokine TL1A to induce prodigious amounts of IL-9 secretion by ILC2s. Unbiased global proteomic analyses revealed that IL-9 was the most-induced protein in ILC2s treated with IL-33 and TL1A. More than 99% of ILC2s expressed IL-9 intracellularly after co-stimulation with IL-33 and TL1A. ILC2s are known to produce very large amounts of IL-5 and IL-13, but ILC2s producing such high levels of IL-9 have not been described previously. Interestingly, IL-9 production by ILC2s was transient and associated with a phenotypic shift characterized by upregulation of IRF4, pSTAT5 and downregulation of ICOS, KLRG1 as well as master transcription factor GATA-3 and its target genes (IL-9R, ST2).
Lastly, we detected an expansion of IL-9high ILC2s in the lungs of mice after IL-33/TL1A inhalation and adoptive transfer experiments revealed that IL-9high ILC2s are potent inducers of allergic airway inflammation in vivo. Thus, our study supports the rising plasticity of ILC2s and their critical role in the induction of type-2 inflammatory responses in the lungs.
T-bet expression is fine-tuned for balancing IFN-γ-producing Th1 and Tfh cell differentiation and IgG2a(c) production
Authors: Fang Difeng , Zheng Mingzhu , Mao Kairui , Reiner Steven L , Sher Alan , Zhu Jinfang (Jeff) 
Published: January 2023

Abstract
T follicular helper (Tfh) cells and cytokine IFN-γ are involved in Ig class switching in B cells to produce IgG2a(c). Tfh cells mainly produce IL-21 as their signature cytokine, and several studies have also shown that some Tfh cells are capable of expressing Th1 signature cytokine IFN-γ without expressing the Th1 master transcription factor T-bet. In Toxoplasma gondii peptide AS15/Complete Freund’s Adjuvant (CFA)-induced immune response, we found antigen AS15-specific IgG2a(c) were abolished in CD4-Cre-mediated T-bet-deficient mice. By using T-bet-fate-mapping mouse strains, we reported that transient expression of T-bet epigenetically imprints the Ifng locus for cytokine production in a subset of Tfh cells; In germinal centers, multi-color tissue imaging revealed that the ex-T-bet Tfh cells express IFN-γ in situ; IFN-γ-expressing Tfh cells are absent in T-bet-deficient mice. More interestingly, we noticed that IFN-γ-expressing Tfh cells were fully present in mice with T-bet deletion at late stages of T cell differentiation, and antigen AS15-specific IgG2a(c) were even further up-regulated in such “T-bet late knockout” mice. Taken together, our findings demonstrate that IFN-γ expressed by T cells plays a dominant role in antibody IgG2a(c) production; T-bet expression is fine-tuned as spatiotemporal manner for balancing Th1 and Tfh cell differentiation in vivo and regulating the strength of antibody production.
Id2 is necessary for differentiation and survival of adipose-resident Tregs
Authors: Frias Adolfo B , Hyzny Eric J , Beppu Lisa Y , Buechel Heather M , D’Cruz Louise M 
Published: January 2023

Abstract
Adipose regulatory T cells (aTregs) have emerged as critical cells for the control of local and systemic inflammation that is linked to health problems including type II diabetes and cardiovascular disease. While a number of factors are known to be required for aTreg survival, in this study we show a distinctive role for the transcriptional regulator Id2 in the maintenance and differentiation of aTregs. Here we show Id2 is highly expressed in aTregs compared to high Id3 expression in lymphoid Tregs. Treg-specific loss of Id2 resulted in a substantial decrease in the frequency of aTregs and a decrease in aTreg surface and intracellular markers while Tregs in the spleen and lymph nodes were unaffected. Gene expression analysis revealed that Id2 expression was essential for the survival of aTregs and loss of Id2 increased cell death in aTregs due to increased Fas expression. Surprisingly, we observed higher Hif1α expression in WT aTregs that diminished with loss of Id2. Thus, we reveal an unexpected role for Id2 in regulating survival of adipose-resident Tregs, critical cells for control of obesity-associated local and systemic inflammation.
D-amino acid oxidase contributes IgA nephropathy through regulation of IgA production in intestinal mucosa
Authors: Suzuki Masataka , Sujino Tomohisa , Goto Motohito , Takahashi Riichi , Ito Mamoru , Sasabe Jumpei , Yasui Masato 
Published: January 2023

Abstract
Gut mucosa protects our body against threats of bacteria using antimicrobial proteins such as defensins, cathelicidins or lysozyme. D-amino acid oxidase (DAO) is one of the antimicrobial protein that is released from goblet cells into lumen and catabolize D-amino acids, which is essential for almost all bacteria, into antibacterial agent; H2O2.
IgA is major immunoglobulin in gut. Maturation of B cells into IgA-secreting plasma cells are lead through two pathways; T cell dependent or independent mechanism. A number of reports indicated the accumulation of systemic IgA in the animals lacking antimicrobial proteins. However, the mechanism how loss of innate immunity affects acquired immunity is yet to be clarified.
We found that mouse lacking DAO activity showed accumulation of systemic IgA. Analysis of intestinal microbiota in DAO null mice revealed increased amount of specific bacteria that stimulated IgA production. Crossbreeding experiment between DAO null mice and T cell receptor double knockout (Tcrb−/−, Tcrd−/−) mice showed that the hyper production of IgA is caused by T cell dependent B cell activation.
IgA nephropathy (IgAN) occasionally causes chronic kidney impairment. Recent genome wide association analysis indicated the linkage between IgAN and genes associated with mucosal immunity. We found that high IgA (HIGA) mouse, an animal model of IgAN, lacked DAO activity, and that high plasma IgA in HIGA mice is dependent on intestinal microbiota. These results indicated that loss of DAO activity involves in pathogenesis of IgAN through disturbance of mucosal immunity.
Use of recombinant influenza vaccines expressing exogenous miRNAs for increased vaccine production
Authors: Stambas John , Ye Siying , Bakre Abhijeet , Edwards Sarah , Dlugolenski Daniel , Tripp Ralph A 
Published: January 2023

Abstract
Vaccination remains the most effective means by which influenza virus infection can be controlled. Traditionally vaccine production has focused on egg-based technology. Increasingly cell culture-based vaccine production is being used to improve preparedness. Cell cultured based production methods offer significant advantages over traditional egg-based strategies by allowing (i) rapid scale-up (ii) elimination of allergic egg components and (iii) circumvention of issues relating to egg-adaption. Our aim was to improve influenza vaccines and preparedness through the establishment of mammalian cell (Vero)-based vaccines using plasmid-based recombinant flu constructs expressing exogenous miRNA19 (flu-miR19), a regulator of key host anti-viral pathways that has previously been shown to modulate influenza virus replication. We successfully rescued recombinant influenza viruses expressing miR19 using reverse genetics; validated expression of miR19 and showed increased virus replication in vitro that corresponded with increased HA titres and matrix gene expression. Knockdown of miR19 host target genes was confirmed through qRT-PCR to validate the mechanism of action. This new influenza vaccine platform is scalable and can be merged with existing production technology to offer rapid clinical translation and improve vaccine quality and production times. In summary, silencing of anti-viral host genes using a flu-miR resulted in an increase of influenza vaccine and HA antigen yield. We now plan to further augment antigen yield by propagating these viruses in enhanced Vero cell vaccine substrates.
Quantifying Immune Effector Molecules from Mouse Leukocytes with Flow-Based Multiplex Assay Panels
Authors: Sun Binggang , Rughwani Priyanka , Chow Kay , Zhao Amy , Lehmann Jason , Jiang Weiping , Ji Shaoquan 
Published: January 2023

Abstract
Exposure to foreign material triggers host immune responses, which are primarily mediated by leukocytes including macrophages, neutrophils, dendritic cells, NK cells, T cells and B cells. These cells produce an array of soluble factors which coordinate host defense, tissue repair, and immunoregulatory functions. In addition, these cells and the factors they produce are also critically involved in various diseases including infection, autoimmune disorders, asthma, diabetes, and cancer. Therefore, quantification of these molecules in a cell type-specific context will help achieve in-depth understanding of the immune responses and disease processes.
We have developed multiplex assay panels targeting specific mouse immune cells (CD4+ T helper cells, Macrophages/Microglia and B cells), using fluorescence–encoded beads that are suitable for use on common lab flow cytometers. Each panel allows for simultaneously quantification of 13 related analytes as shown in the table below.
Cell TypePanel TargetsT helper cellsIL-2, 4, 5, 6, 9, 10, 13, 17A, 17F, 21, 22, IFN-γ, TNF-αMacrophages/MicrogliaIL-1β, 6, 10, 12p40, 12p70, 18, 23, TNF-α, CCL22/MDC, CCL17/TARC, CXCL1/KC, G-CSF, TGF-β1B cellsIL-2, 4, 6, 10, 12p70, 13, 17A, IFN-γ, TNF-α, BAFF, BCMA, sCD40 ligand, TGF-β1
Each antibody pair was carefully optimized for assay specificity, sensitivity, accuracy and reproducibility. In addition, these panels have been validated by detecting expected expression level changes of analytes in biological samples cultured under various stimulation conditions. These high quality, low cost and easy to use panels will be useful tools to the study of specific cells involved in innate and adaptive immune responses and the relevant diseases.
20,000 layers under the model: Bayesian neural networks improve the robustness of human PBMC classifications using immunologist encoded bias
Authors: Velazquez-palafox Miguel Angel , Taylor Ian James , Spidlen Josef , Lai Janice , Halpert Richard , Stadnisky Michael 
Published: January 2023

Abstract
Accurate classification of immunological subsets is a cornerstone of routine assays in basic research and the clinic. This process generates terabytes of data for analysis, creating bottlenecks in reproducibility and feasibility. Advances in machine learning, specifically in deep learning, have dramatically expanded solutions to address this issue with higher accuracy and performance. Here we applied a supervised deep learning approach to guide cell classification of canonical human PBMC immune cell types from oligo-tagged antibodies and sequencing datasets. Utilizing Bayesian neural networks, we leveraged probabilities to encode scientist knowledge into the models whilst extrapolating to new datasets. The developed models produced higher F1 scores when compared to trained immunologists, data scientists, and traditional machine learning techniques. We demonstrate robust cell-type annotation over both validation and test PBMC data sets with over 95% accuracy. Furthermore, this architecture revealed the weighted relationships between the inputs and cell type predictions, giving insight into the driver genes and cell markers.
New generation CD90.1 and CD90.2 MicroBeads: fast isolation of total of adoptively transferred T cells from mouse spleen
Authors: Evaristo Cesar , Pitzka Christina , Pietz Nicole , Foerster-Marniok Anna , Dose Christian , Richter Anne 
Published: January 2023

Abstract
T cells play a central role in immunity to pathogens and tumors, but also in autoimmunity. T cell research has generated knowledge that led to multiple clinical breakthroughs, such as the generation of T cell vaccines and tumor immunotherapy approaches. However, additional work is required to fully understand T cell biology, harness their therapeutic potential and control immunopathologies. Cutting edge experimental protocols that interrogate and manipulate T cell biology often require prior purification of T cell populations. Strategies to simplify and accelerate T cell purification are highly desirable to save time, reduce bias and allow complex experiments to be performed.
We established workflows combining automated tissue dissociation with T cell isolation. We developed new CD90.1, and CD90.2 T cell specific enrichment reagents that significantly accelerated the isolation protocol. These reagents could be used to isolate total T cells, or rare CD90.1+ or CD90.2+ cells from adoptive transfer models. Importantly, culture of isolated T cells did not induce activation as measured by CD69 or CD25 upregulation, as compared to controls. Furthermore, magnetic isolation did not affect proliferation or cytokine expression following in vitro stimulation. Finally, T cell isolation could be fully automated and multiple samples could be processed in parallel.
Our new workflow greatly reduces time of downstream analysis while preserving cell phenotype and functional properties. We believe these innovative tools significantly shorten time-consuming experiments and can be used to increase reproducibility and the quality of data obtained in T cell research.
Rapid modification of specificity and affinity of anti-influenza VHH antibody using two in vitro evolution methods
Authors: Kishimoto Hidehiro , Yoshida Maiko , Tsukahara Narutoshi , Matsubara Toshiyuki , Nakayama Hideorshi , Murakami Akikazu 
Published: January 2023

Abstract
Heavy-chain antibodies (HCAbs) were discovered in camelids in 1990s and the Ag-binding fragment of the HCAbs consists of a single domain known as a variable domain of the heavy chain of HCAbs (VHH). Because of the many favorable properties of VHHs, i.e. small size (12kDa-15kDa), high affinity, high-stability and well expression in microorganisms, they have become widely used in research and are beginning to show commercial potential. Most of the VHHs have been isolated from recombinant VHH phage display libraries from immunized camels or llamas and their successive panning has been the preferred method for the isolation of high-affinity specific binders, however, the procedure is time-consuming and costly. Therefore, we constructed semi-synthetic alpaca VHH phage display libraries having more than 20 billion repertoires. From the library, Ag specific VHH clones can be isolated within 10 days, and we have obtained hundreds of VHH clones against more than 30 different Ags. One of the anti-influenza HA (H1N1) VHH clone has neutralization activity. Unfortunately, the clone only binds to H1 sub-type of hemagglutinin. Therefore, we optimized two kind of in vitro evolutional methods to modify the specificity and affinity of the anti-H1 VHH clone. Random mutagenesis is commonly used for evolution of antibodies using the phage display method, but it is very difficult to obtain clones with greatly increased affinity. We have already constructed a rapid affinity improvement method using phage display system, CDR3 preservation method, thus we combined these two methods to create new rapid modification system of VHH antibodies. Here, we show an example in which specificity was changed from influenza H1 to H5, and further improved affinity.
Zika virus detection using a flow cytometry platform
Authors: Wood Steven C 
Published: January 2023

Abstract
Zika is an arthropod-borne virus related to other Flaviviridae (FV) members such as West Nile Virus, Yellow Fever Virus Japanese Encephalitis Virus and Dengue Virus All these FV infections can develop into indistinguishable clinical symptoms such as headaches, fever, body aches, and joint pains. However, in utero exposure to Zika results in microcephaly, fetal demise and Guillain–Barré syn-drome. These devastating complications necessitated the development of a specific and sensitive assay that distinguishes Zika infection from those caused by other FV. Development of a Zika specific immune-detection assay is challenging due to the presence of cross reactive antibodies. Therefore, we developed a high throughput Zika detection assay using a flow cytometry platform and Zika antigens as reference materials. Sera from Zika positive and negative patients were diluted 100, 1,000 and 10,000 fold and used for the initial standardization experiments. Minimal background fluorescence was detected at 10,000-fold dilution of sera, and therefore the assay was standardized at these optimal conditions. Flow cytometric analysis revealed the presence of anti-Zika antibodies in Zika patients and out of 10 patient samples tested, 9 were confirmed to be Zika positive. The ability of this flow cytometry based assay to detect anti-Zika antibodies was also confirmed using sera samples collected from Zika Ns1 and Env protein immunized mice. In summary, we report the development of a novel flow cytometry based Zika detection method that can be used for Zika counter measures. The rapid turnaround time of flow based assays as well as their ability to detect and distinguish antibody subtypes and antigens will improve diagnostic accuracy of Zika detection.
High Throughput pMHC-I Multimer Library Production Using Chaperone-Mediated Peptide Exchange
Authors: Overall Sarah , Toor Jugmohit S , Yarmarkovich Mark , Hao Stephanie , Nguyen Son , Sada-Japp Alberto , Betts Michael R , Maris John M , Smibert Peter , Sgourakis Nikolaos G 
Published: January 2023

Abstract
The development of pMHC-multimer technologies has been indispensable for the characterisation of T cell responses to individual viral and tumor peptide antigens. Given the large variety of T cell receptors expressed in polyclonal repertoires, peptide exchange technologies are becoming increasingly important in providing a high throughput means of generating pMHC-multimer libraries, containing a wide range of antigens to probe such repertoires. Here we present a simple method for the isolation of empty MHC-I molecules which can be readily used for high throughput multimer library preparation. We utilise the chaperone TAPBPR, which binds MHC-I molecules and stabilises them in a peptide-receptive conformation, to isolate empty MHC-I molecules through the combination of placeholder peptides and small molecule competitors. We demonstrate that empty H-2Dd:TAPBPR and HLA-A*02:01:TAPBPR complexes can be stored long term and subsequently loaded with peptides of choice in a high throughput fashion to afford pMHC-multimer libraries. The resulting pMHC-multimers show equivalent flow cytometric properties to multimers made from MHC-I molecules refolded in vitro with synthetic peptides and are significantly improved relative to multimers prepared using peptide exchange of conditional ligands. Using this simple system to generate a library of oligonucleotide-labeled pMHC-multimers, combined with our recently described multi-modal cellular indexing technology (ECCITE-seq), we can identify distinct T cell receptor sequences present in polyclonal repertoires, together with their antigen specificities.
A universal planar assay format for high sensitivity cytokine quantification in human serum and plasma
Authors: Lambert Jeremy , Mendes Muriel , Yan Yu-xin , Ball Andrew 
Published: January 2023

Abstract
Quantifying prospective biomarkers of immune signaling in biological samples requires high assay sensitivity. The Simoa Planar Array provides greater sensitivity than ELISA, but has a finite assay menu. This study aimed to develop a universal ‘homebrew’ planar immunoassay format for development of high sensitivity assays for any analyte, by using an anchor antibody/peptide tag system to develop a universal capture plate.
Anchor antibody specific for a peptide tag was microprinted in microwell plates. The peptide tag was conjugated to analyte-specific capture antibodies via maleimide chemistry, enabling high affinity binding of capture to anchor antibody. Bound sample antigen was sandwiched between the peptide-tagged capture and biotinylated detector antibodies; chemiluminescent detection was used to quantify signal.
This ‘homebrew’ approach successfully detected sub picomolar levels of cytokines and chemokines in human or mouse serum and plasma, including IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-22, ITAC, MIP1a and MIP3b. When compared to commercially available planar assays the universal planar assay format provided comparable LLOQ and LOD with sample correlation (R2) &gt;0.97 for all analytes tested. The universal planar assay was used to successfully screen multiple antibody pairs for human and mouse biomarkers and to develop robust assays using mouse, rabbit or goat monoclonal and polyclonal antibodies.
This universal planar homebrew assay format combines the accuracy, reproducibility and high sensitivity of Simoa Planar Array technology with total analyte flexibility, and provides assay developers a simple and versatile method to rapidly develop robust high-sensitivity assays for any analyte of their choice.