bluuebunny/crossref_metadata_2025_split huggingface dataset:
Obesity-induced dysregulation of a unique subset of Tregs promotes skin inflammation
Authors: Li Chaoran , Sivasami Pulavendran , Elkins Cody , Goss Kyndal , Diaz-Saldana Pamela P , Peng Amy , Hamersky Michael , Bae Jennifer , Pollack Brian P , Horwitz Edwin M , Scharer Christopher D , Seldin Lindsey 
Published: August 2023

Abstract
Obesity is associated with increased skin inflammation and is a major risk factor for psoriasis, raising the question of how obesity disrupts the regulatory mechanisms that keep skin inflammation in check at steady state. We found that skin was enriched with a unique subset of CD4+ Foxp3+ regulatory T cells (Tregs), which is critical to limit IL-17A-mediated psoriatic inflammation. Diet-induced obesity, however, resulted in a significant reduction of this subset of skin Tregs and a corresponding loss of control over IL-17A-mediated inflammation. Mechanistically, this specific skin Treg population preferentially took up elevated levels of long-chain saturated fatty acids during obesity, which led to cellular lipotoxicity and mitochondrial dysfunction. Harnessing the anti-inflammatory property of this unique subset of skin Tregs could have therapeutic potential for obesity-associated inflammatory skin diseases.
Supported from grants from NIH/NIDDK (1R01DK128061), National Psoriasis Foundation, and Emory University Research Committee (URC).
Lung injury induces preferential expansion of Foxp3+ regulatory T cells over conventional T cells in self-antigen specific CD4+ T cell repertoire
Authors: Shin Daniel Seong-Joo , Moon James J 
Published: August 2023

Abstract
Self antigen-specific T cells are prevalent in the mature adaptive immune repertoire. At steady state, these T cells are regulated by various mechanisms of peripheral tolerance. Characterizing the activation of these self antigen-specific T cells during inflammation may shed crucial insights into the pathogenesis of autoimmunity. Utilizing an experimental animal model where mice express three model T cell epitopes (2W, gp66, and OVA) in a tissue restricted manner, we detect the exclusive expansion of autoreactive regulatory T cells (Tregs) following acute lung injury that is dependent on recognition of their cognate self antigen and IL-2 signaling. Conversely, conventional T cells of the same self antigen-specificity did not expand during inflammation, even following the depletion of endogenous Tregs. Therefore, our findings suggest that the self-antigen specific T cell repertoire may serve a regulatory function during acute tissue damage to regulate the ongoing immune response and limit the possibility of autoimmunity.
IEDB-3D 2.0: Enabling visualization of epitopes on the 3D structure of the corresponding antigens
Authors: De Almeida Mendes Marcus F , Mahita Jarjapu , Blazeska Nina , Wheeler Daniel , Ha Brendan , Greenbaum Jason , Wang Jiyao , Shackelford Deborah , Sette Alessandro , Peters Bjoern 
Published: August 2023

Abstract
The Immune Epitope Database and Analysis Resource (IEDB) contains information related to antibodies, T cells, and MHC across a broad spectrum of research fields (infectious diseases, cancer, allergy, autoimmunity, and transplantation). A critical component of this information is three-dimensional structural data on T cell receptors (TCRs), antibodies, and pairwise residue interactions between immune receptors and antigens, which we refer to as IEDB-3D. Analysis using atomic models helps to understand the intricate interactions between immune receptor and epitopes. Here, we present IEDB-3D 2.0, which incorporates a complete overhaul of how we obtain and present 3D structural data from PDB and AlphaFold database. We implemented a new molecule visualizer, iCn3D, to replace outdated java-based technology. In addition, we have designed a new epitope mapping system that matches each epitope available in the IEDB with its antigen structural data. Lastly, we integrated our epitope mapping system with immunogenic data retrieved from the IEDB’s ImmunomeBrowser, which now can be used to highlight immunogenic regions of an antigen directly in iCn3D. Overall, the IEDB-3D 2.0 provides an updated tool platform to visualize epitope data cataloged in the IEDB.
Research reported in this publication was supported by the National Institutes of Health contract 75N93019C00001 and grant U24CA248138.
An integrated reagent and multimodal analysis workflow to enrich and characterize peptide-specific CD8+T cells
Authors: Adams Bruce A , Shahi Payam , Reyes Daniel , Maheshwari Shamoni , Mousavi Nima , Krishnan Sreenath , Ramen Nandhini , Tsai FuNien , Kumar Poornasree , Finnegan Peter , Fernandes Sunjay , Bali Gunmeet Kaur , Terry Jessica 
Published: August 2023

Abstract
Immune responses to disease are complex and the only way to understand such a response is to consider the behavior of multiple different cell types across both the innate and adaptive branches of the immune system. We developed Barcode Enabled Antigen Mapping (BEAM) technology to enable easy enrichment of antigen-specific cells. This technology is fully integrated with the 10x Genomics Chromium Single Cell Immune Profiling Solution to allow multimodal evaluation of immune cells including full-length, paired sequences of T and B cell receptor genes while simultaneously screening their specificity for a set of genes. Our antigen-specific TCR workflow (BEAM-T) identifies antigen-specific CD8+T cells. We demonstrate the assay’s performance with spike-in experiments using various percentages of HLA-A*02:01-restricted peptide-stimulated T cells (targeted mixture being 40% T cells with specificity for cytomegalovirus peptide, 10% T cells with specificity for flu peptide and 0–5% anti-SARS-CoV-2 T cells) into a background of human PBMCs. Cells were labeled with a panel of five viral pathogen peptide-loaded multimers, plus a HLA-A*02:01 negative control peptide to assess antigen specificity and were then sorted for CD8+PE+ T cells. We demonstrated that the major VDJ-T clonotypes identified for the SARS-CoV2 cells were retained, albeit with roughly proportionally fewer cells, as the percentage of spiked cells decreased. We further feature the robustness of the protocol with various sample types and models including H2Kb-restricted OT-1 transgenic mouse splenocytes, as well as healthy human donor PBMCs and dissociated tumor cells to demonstrate the utility of an integrated reagent-to-data analysis offering across a range of samples.
BD Pharmingen™ Leukocyte Blocking Buffer reduces nonspecific binding of cyanine-like dye conjugates to monocytes and other leukocytes
Authors: Wei Ai-Li , Gao Guo-Jian , Ernst David , Cantor Joseph M , Li Li , Verma Shilpi 
Published: August 2023

Abstract
Nonspecific binding of monocytes to certain cyanine-like dye conjugated antibodies that are commonly used in flow cytometry for cell surface staining is well known. Although lesser in extent than monocytes, other leukocyte subsets can also exhibit nonspecific binding to cyanine-like dye conjugates. This poses a challenge for multicolor flow cytometric analysis utilizing these cyanine-like dye conjugates as dye-mediated nonspecific binding can mask the real expression. Here we demonstrate that the BD Pharmingen™ Leukocyte Blocking Buffer, a blocking buffer solution, eliminates nonspecific binding of cyanine-like dye conjugates to monocytes and other leukocyte populations. In this study we show that human whole blood stained with PE-Cy5, PE-Cy7, APC-Cy7, APC-H7, PE-CF594 and other tandem-dye conjugated antibodies in the presence of this new buffer exhibits the appropriate staining profile of these antibodies to monocytes and other leukocyte subsets. We also observed that nonspecific binding of cyanine-like dye conjugated antibodies to myeloid and lymphoid subsets in mouse lymphoid tissues was diminished. BD Pharmingen™ Leukocyte Blocking Buffer, while reducing nonspecific cyanine-like dye conjugates binding, did not impact the desirable antibody-mediated specific staining. Our data also show that cell viability is not impacted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD and Pharmingen are trademarks of Becton, Dickinson and Company or its affiliates. © 2023 BD. All rights reserved. NPM-0263 (v1.0) 1222
CF is a trademark of Biotium, Inc. Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
A lymph node slice culture model to characterize T cell activation dynamics and anti-viral responses in human tissue
Authors: George Alex , Davis-Porada Julia , Weng Shannon , Zhao Wenting , Farber Donna L , Sims Peter 
Published: August 2023

Abstract
T cell activation in the lymph node (LN) is critical to initiating adaptive immune responses to vaccination, tumors, and infection. We previously demonstrated that T cells from human tissue sites (blood, LNs, bone marrow, lung) undergo conserved cell state transitions during activation. However, these studies were performed in suspension culture from dissociated tissue, depleting the spatial and signaling contributions of the tissue microenvironment. To address this, we developed techniques for culture and single-cell analysis of human LN slices, which preserve tissue architecture, local signaling, and physiological cell frequencies. Single-cell RNA-seq (scRNA-seq) of matched LN suspension and slice cultures stimulated with anti-CD3/28 antibody tetramers revealed distinct activation patterns. While anti-CD3/28 stimulation is commonly used to study T cell activation, this reagent circumvents antigen presentation and may not fully recapitulate antigen-mediated signaling. Conversely, antigen-dependent stimulation with superantigen mimics require MHCII/TCR binding and may benefit from the intact microenvironment in slice culture. We discovered a distinct metabolic intermediate through scRNA-seq analysis of superantigen-stimulated LN suspension and slice cultures that is not observed with anti-CD3/28 stimulation. Overall, these studies demonstrate the utility of analyzing T cell responses in intact tissue and comparative analysis of distinct mechanisms of activation.
1T32GM145440-01 (MSTP Training Grant) U19AI128949 (NIAID)
A 47-marker immune profiling flow cytometry assay to enable comprehensive antigen-specific immune analysis
Authors: Cohen Michael J , Yao Huihui , Li Stephen , Tracey Lauren , Loh Christina 
Published: August 2023

Abstract
The complex nature of the immune system requires deep interrogation at the single-cell level. Flow cytometry using CyTOF® technology employs antibodies tagged with isotopically enriched metals instead of fluorophores, resulting in discrete signals that facilitate highly multiparametric characterization on a single-cell level. CyTOF technology enables analysis of over 50 parameters simultaneously without the impediments of spectral overlap or autofluorescence, allowing for a reliable evaluation of immune responses.
The Maxpar® Direct™ Immune Profiling Assay™ is a pre-titrated, dried-down, and validated 30-marker antibody cocktail for single-tube staining of human whole blood or PBMC and acquisition on CyTOF systems. Using the assay and Maxpar Pathsetter™ software, stained samples are automatically resolved into 37 immune populations. In this study, we highlight the customizability of the Maxpar® Direct panel by adding an array of 17 surface and intracellular parameters with the Maxpar Direct™ T Cell Activation Expansion Panel and live-cell barcoding. The simultaneous detection of key surface and intracellular markers in this expanded panel enabled comprehensive analysis of immune cell activation and antigen-specific recall responses to PBMC stimulated with a pool of immunodominant microbial peptides.
Taken together, this 47-marker panel and automated analysis unlocked deeper immunophenotyping and functional profiling of activated immune cells and provided insight into the cell-mediated adaptive immune response to foreign targets. Such phenomena are hallmarks for research on infection, vaccine development, and immunotherapy.
For Research Use Only. Not for use in diagnostic procedures.
Unraveling T cell mitochondrial dynamics using imaging flow cytometry
Authors: D’Cruz Louise M , Shi Xiaoshan , Widmann Stephanie J , Tyznik Aaron J 
Published: August 2023

Abstract
Mitochondria are highly mobile, dynamic subcellular organelles that mediate energy generation in mammalian cells. In response to changes in energy and stress, mitochondria can either join or divide, a process known as fusion or fission, respectively. Mitochondrial morphology plays a critical role in the metabolic state of T cells and can affect T cell function in health and disease. Despite increased interest in mitochondria dynamics, a robust and efficient method to simultaneously image mitochondria morphology, analyze T cell phenotypes and sort cells has not been feasible. Here, we used the state-of-the-art BD CellView™ Image Technology on the BD FACSDiscover™ S8 Cell Sorter to detect changes in mitochondria morphology with high resolution and profile T cell phenotypes associated with specific mitochondrial shapes. With the imaging capabilities of the BD FACSDiscover™ S8 System, we visualized live mitochondria and nuclei using three-color staining in a high-throughput manner. We differentiated effector T cells in vitro and imaged increased fission-like mitochondria in these cells. With the cell sorting feature of the BD FACSDiscover™ S8 System, we sorted effector T cells based on their cell surface phenotype and distinct mitochondrial characteristics and evaluated their mitochondrial gene signature. Thus, we provide an advanced and comprehensive workflow to unravel mitochondrial dynamics and isolate cells with a particular phenotype for downstream in-depth characterization enabled by the BD FACSDiscover™ S8 System. As many cellular processes are linked to mitochondrial dynamics, this technique provides a powerful tool to understand how this critical organelle is regulated in a variety of human diseases.
Development of a 40+ color full spectrum flow cytometry panel for in-depth immunophenotyping of chief cell subsets in non-human primate peripheral blood mononuclear cells
Authors: Fears Alyssa , Geisbert Thomas , Woolsey Courtney 
Published: August 2023

Abstract
Leveraging full-spectrum technology, we designed and optimized a 40+ color flow cytometry panel for the analysis of non-human primate peripheral blood mononuclear cells (PBMCs). Non-human primate models of disease allow for the closest approximation of human outcomes to vaccination, disease, and treatment due to similarities in physiological host responses, which increase predictive accuracy. This first panel of its kind achieves comprehensive profiling of immune subpopulations by the inclusion of surface, intracellular, and cytokine markers representative of differentiation, activation, functionality, and proliferation to deeply interrogate the immune response to infectious disease and vaccination. The optimized panel is additionally appropriate for the discovery of new immune phenotypes important for protection against pathogens. After initial optimization for use with cynomolgus macaque (Macaca fascicularis) and rhesus macaque (Macaca mulatta) PBMCs, the panel was further adapted for use with African green monkey (Chlorocebus aethiops) PBMCs to allow for cross-comparison between three frequently implemented non-human primate models of infectious disease.
This study was supported by the US Army Medical Research Acquisition Activity contract number W81XWH1910027 to TWG and Department of Health and Human Services, National Institutes of Health grant number UC7AI094660 for BSL-4 operations support of the Galveston National Laboratory.
Spatially resolved immune receptor repertoires from Visium transcriptomic libraries
Authors: Montagne Janelle Marie , Sidiropoulos Dimitrios N , Mo Guanglan , Zhu Qingfeng , Anders Robert A , Zheng Lei , Jaffee Elizabeth M , Larman H Benjamin , Fertig Elana J , Kagohara Luciane T 
Published: August 2023

Abstract
Recent advances have enabled the integration of T cell receptor (TCR) sequencing into spatial transcriptomics. Current approaches yield critical information about in situ T cell responses directly from relevant tissues, yet are largely limited to alpha/beta TCRs, leaving the clonal dynamics of B cells and gamma/delta T cells underexplored. We therefore modified our previously reported ultra-efficient immune receptor repertoire sequencing (IRR-seq) method, Framework Region 3 AmplifiKation Sequencing (FR3AK-seq), for compatibility with the 10X Genomics Visium spatial transcriptomics platform. Here we report simultaneous acquisition of alpha/beta TCR, gamma/delta TCR, and immunoglobulin (B cell receptor, BCR) heavy/light chain sequences from a single multiplexed FR3AK-seq reaction using Visium transcriptomics-generated cDNA libraries. More specifically, we used the Visium spatial transcriptomics protocol to generate cDNA from serial sections of a lymph node with a metastatic lesion obtained from a patient with pancreatic ductal adenocarcinoma previously treated with immunotherapy. Notably, we detected sequences from all IRR chains as expected from the abundant immune infiltrates within these sections. Furthermore, we found that multiplexed FR3AK-seq yields technically reproducible repertoires, with the TCR beta chain repertoires also comparable to those obtained using a previously published method. We anticipate that this simple extension of the Visium technology will accelerate the simultaneous and comprehensive examination of in situ IRR dynamics beyond alpha/beta T cells.
Decoupling peptide binding from T cell receptor recognition with engineered chimeric MHC-I molecules
Authors: Papadaki Georgia , Ani Omar , Florio Tyler , Young Michael , Danon Julia , Sun Yi , Dersh Devin , Sgourakis Nikolaos 
Published: August 2023

Abstract
Human leucocyte antigens (HLAs) display a repertoire of epitopic peptides on the cell surface for T cell recognition. Amino acid polymorphisms in the peptide-binding groove of different HLA allotypes result in distinct antigenic repertoires, providing a basis for adaptive immunity to emerging pathogens at the population level. T cell receptors (TCRs) recognize pHLA antigens through interactions with both peptide and heavy chain residues. In this work, we use structural data from peptide:MHC-I and pMHC:TCR structures to identify residues important for peptide and/or TCR binding, and we outline a fixed-backbone computational design approach for engineering synthetic molecules that combine peptide binding and TCR recognition surfaces from existing HLA allotypes. X-ray crystallography demonstrates that chimeric molecules bridging divergent HLA alleles can bind selected peptide antigens in a specified backbone conformation. Moreover, in vitro tetramer staining and biophysical binding experiments using chimeric pMHC-I molecules presenting established antigens further demonstrate the requirement of TCR recognition on interactions with HLA framework residues, as opposed to interactions with peptide-centric Chimeric Antigen Receptors (CARs). Our results underscore a novel, structure-guided platform for developing synthetic HLA molecules displaying tumor-associated antigens with modified TCR interaction surfaces, which could further serve as platforms to elicit alloreactive T cell responses against self-antigens that are overexpressed in cancer. Finally, we gain a deeper understanding of the evolution and functional divergence of the different HLA allotypes by demonstrating an example of convergent evolution.
Supported by grants from NIAID (5R01AI143997), NIGMS (5R35GM125034), and NIDDK (5U01DK112217)
CD8+ T-cell memory induced by SARS-CoV-2 mRNA vaccination is maintained by clonal replenishment
Authors: Aoki Hiroyasu , Kitabatake Masahiro , Abe Haruka , Shichino Shigeyuki , Hara Atsushi , Ouji-Sageshima Noriko , Ito Toshihiro , Matsushima Kouji , Ueha Satoshi 
Published: August 2023

Abstract
mRNA vaccines against the Spike glycoprotein of severe acute respiratory syndrome type 2 coronavirus (SARS-CoV-2) elicit strong CD8+ T-cell responses. However, in repetitive vaccination, it is unknown whether T-cell clones induced by the first dose respond repeatedly or new clones are mobilized and respond after each dose. Here, we temporally tracked the CD8+ T-cell repertoire in individuals who received three doses of the BNT162b2 mRNA vaccine. CD8+ T-cell clones that expanded after the first, second, or third dose were identified, and their kinetic profiles throughout the vaccination were analyzed. The extent of clonal expansion after the first vaccination was significantly less than that after the second or third vaccination. After the second vaccination, clones that had expanded after the first dose (“first responders”) were less responsive, while a new set of clones (“second responders”) greatly expanded and surpassed the first responders. After the third vaccination, the second responders expanded again, while the first responders did not. Moreover, we observed expansion of another set of clones that had not responded to the previous doses (“third responders”). Single-cell mRNA/protein/TCR analysis after the second vaccination revealed that the first-responder clones exhibited a terminally differentiated phenotype, whereas the second-responder clones exhibited an actively proliferating phenotype. These results suggest that CD8+ T-cell memory induced by repetitive mRNA vaccination is maintained by the mobilization of newly generated clones with proliferative potential.
This work was supported by Japan Society for the Promotion of Science (JSPS) under Grant Number 17929397 and 20281832, and Japan Agency for Medical Research and Development (AMED) under Grant Number JP22fk0310509, JP22ama221306, and P21gm6210025.
The Comparison of the Duration of Immunity Induced by Pertussis Vaccines and Infection in a Baboon Model
Authors: Wang Yihui , Merkel Tod 
Published: August 2023

Abstract
Whooping cough is a highly contagious, acute respiratory illness caused by the Gram-negative bacteria, Bordetella pertussis. Disease is often mild in older children and adults but can be severe or fatal in young children and infants. It is endemic worldwide and a major cause of vaccine-preventable deaths despite high vaccine coverage rate. A steady increase in the number of reported pertussis cases has been evident over the last 20 years. The apparent correlation of this resurgence and the replacement of whole-cell (wP) vaccines with acellular (aP) vaccines suggests the current aP vaccines may not control pertussis as well as the wP vaccines they replaced. Some evidence indicates the aP vaccines may induce protection with shorter duration of immunity relative to wP vaccines.
There is increased interest in developing next-generation vaccines that induce longer lasting protection. However, immunological correlates predicting durable and protective immunity remain unknown. Studying duration of immunity in a human population is problematic due to potential asymptomatic exposures and the difficulty in sample accessibility. In an ongoing, long-term study, using a baboon model we are comparing the duration of immunity and immunological correlates of protection in a controlled population comparing aP, wP vaccines and natural infection. Extensive sampling was undertaken around the vaccinations to characterize the vaccine-mediated immune responses. Animals enrolled in the study will be followed for years with sampling twice yearly. With strict control of Bordetella exposure, the data generated from the study will provide a clearer understanding and a basis of comparison for the duration of aP, wP, and infection-induced immunity.