bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differentiation and dynamics of circulating and tissue-resident memory CD8 T cells in the lung
Authors: Anthony Scott M , Harty John T 
Published: January 2023

Abstract
Memory CD8 T cells are capable of providing robust heterosubtypic immunity to Influenza. In contrast to other tissues, relatively little is known about the differentiation and localization of the memory CD8 T cell subsets within the lung. We utilized intravital 2-photon microscopy of live mice to examine the real-time dynamics of circulating and/or resident memory (Trms) CD8 T cells in the lung. LCMV-induced transgenic P14 CD8 memory T cells, which are exclusively circulating, were highly motile, while Influenza-experienced P14s, which are comprised of both circulating and resident memory CD8 T cells, exhibited heterogenous motility patterns including both highly motile cells and those exhibiting a slow to moderate motility pattern. Selective depletion of circulating memory CD8 T cells resulted in the loss of highly motile cells, suggesting that lung CD8 Trms exhibit slow to moderate motility. Intranasal administration of cognate peptide or recombinant Influenza virus infection induced the arrest of a large number of LCMV-experienced circulating P14s in the lung shortly after administration in an antigen-specific manner. Interestingly, within 12–48 hours post stimulation, the majority of lung-residing LCMV-experienced circulating P14s became un-arrested and began a moderate scanning motility phenotype that persisted in a substantial fraction of P14s and correlated with the generation of secondary memory CD103+ Trms in the lung. Overall, these data shed light into the differentiation and dynamics of circulating and resident CD8 memory T cells in the lung.
Small Intestinal Cytotoxic CD4 T Cells: Novel Mechanisms of Development and Unique Protective Capability
Authors: Chen Angeline , Iwaya Hitoshi , Thiault Nicolas , Cheroutre Hilde 
Published: January 2023

Abstract
Conventional CD4 T helper T cells convert to CD4 cytotoxic T lymphocytes (CTL) in the small intestinal epithelium during steady state, but the mechanism and antigens that drive their generation and purpose(s) at this barrier are largely unknown. The unique localization of mucosal CD4 CTL to the small intestinal epithelium suggests local non-pathogen antigens and organ-specific properties that direct CD4 CTL to develop from CD4 T helper (Th) cells.
Similar to traditional antigen presenting cells (APCs), intestinal epithelial cells (IECs) express MHC class II (MHCII), indicating a possible influence over nearby CD4 T cells. In addition, IECs are constantly processing luminal contents and therefore are likely to present non-pathogen-derived antigens (e.g. diet or commensal bacteria-derived) to CD4 T cells at its baso-lateral side. Taken together, we hypothesized and now show that the constant presentation of dietary antigens by IECs in a non-inflammatory environment leads to the development of CD4 CTL in the small intestinal epithelium.
These CD4 CTL accumulate over time and fortify the small intestine as an innate-like defensive arm specifically designed to contain infections while minimizing inflammation at this delicate interface. Many types of immune cells are involved in combating Salmonella infection, but CD4 CTL represent a distinct niche. Here, we demonstrate the protective capacity of CD4 CTL to rapidly and effectively kill infected epithelial cells thereby preventing systemic spread of pathogens as well as excessive immune pathology caused by infiltrating peripheral adaptive immune cells.
A fast and robust protocol for immunomagnetic isolation of human CD45+ leukocytes from samples with variable start frequencies
Authors: Posarac Vesna , Ewen Catherine L. , Jovanovic Vida K. , Hayer Elisha , Kokaji Andy I. , Woodside Steven M. , Eaves Allen C. , Thomas Terry E. 
Published: January 2023

Abstract
The study of immune cell function in non-lymphoid tissue and tumors has emerged as an exciting research area, with the promise to provide novel strategies for the treatment of immune disorders, infectious diseases and cancer. A major challenge in the field is the isolation of leukocytes from tissues due to the complexity of starting samples that can be highly variable in frequency and subset composition. We describe a new 28 minute protocol optimized for the isolation of particle-free human CD45+ leukocytes from varied sample sources. Using the EasySep™ Release Human CD45 Positive Selection Kit, leukocytes are labeled with antibody complexes linked to magnetic particles and separated using an EasySep™ magnet. The isolated cells are then released from particles by resuspension in EasySep™ Release Buffer and a final magnetic separation step. Performance was assessed using human leukocytes spiked into a single cell suspension of mouse thymocytes across a range of frequencies to simulate the variable leukocyte content in tissue and tumor samples. Human CD45+ purities were 95.4 ± 4.3% from start ranges of 0.5–2.0% (n=15) and 99.1 ± 0.7% from start frequencies of ≥10% (n=18). The isolated cells are compatible with further downstream isolation and are functional, as demonstrated by T cell proliferation and IFN-gamma production assays. We have also assessed our CD45+ isolation kit from tissues of humanized NSG mice, expanding the application of our protocol to highly relevant disease and cancer models. The EasySep™ Release Human CD45 Positive Selection Kit provides a robust method for high purity isolation from diverse tissues and starting frequencies, generating particle-free cells ready for downstream evaluation.
Identifying antibodies and T-cell receptors with known specificity in repertoire sequencing data
Authors: Mahajan Swapnil , Vita Randi , Yan Zhen , Nielsen Morten , Sette Alessandro , Peters Bjoern 
Published: January 2023

Abstract
The adaptive immune system in vertebrates has evolved to recognize an immense number of pathogens. B and T cells play important role in the antigen recognition by expressing a substantially diverse repertoire of antigen-specific receptors. The recent advances in the high-throughput immune repertoire sequencing has led to a tremendous increase in the antibody and T-cell receptor information. Identification of specific epitopes recognized by the receptors is vital in analyzing the repertoire sequencing data. Here, we present a tool that examines data from an immune receptor repertoire experiment, and identifies receptors (or highly related variants) that are present in the Immune Epitope Database (IEDB). The IEDB is a free resource which compiles experiments characterizing immune epitopes. IEDB also captures the epitope-specific immune receptor sequences and groups them based on their epitope specificity. IEDB currently has information on over 26,000 receptor groups. Users can provide receptor repertoire sequence data in multiple formats, and specify different CDR sequence identity cutoffs to restrict or broaden the receptor search. We expect that this tool will provide new insights into B and T cell responses captured in the receptor repertoire experiments by identifying specific epitopes, and facilitate development of new strategies for monitoring and treating of infectious, allergic and autoimmune diseases.
MHC clustering regulates selectivity and effectiveness of T-cell responses
Authors: Anikeeva Nadia , Fischer Nicholas O , Blanchette Craig D , Sykulev Yuri 
Published: January 2023

Abstract
Although formation of nano-scale MHC clusters on the cell membrane of antigen-presenting cells has been well documented, the essence of MHC clustering and the role in controlling antigen presentation and T-cell responses have been not systematically studied. This is due to the absence of available approaches to evaluate how the clusters’ size, density and composition of MHC clusters influence TCR engagement and triggering of TCR signaling. We exploited nano-scale discoidal membrane mimetics (nanolipoprotein particles, or NLPs) to capture and present pMHC ligands at various densities and examined the binding of these model membrane clusters to the surface of live human CD8+ T cells and the subsequent triggering of intracellular Ca2+ signaling. The data demonstrate that the proximity of pMHC ligands, high association rate of CD8-MHC interactions, and relatively long lifetime of cognate TCR-pMHC complexes emerge as essential parameters explaining the significance of MHC clustering. Rapid rebinding of CD8 to clustered MHC suggests a dual role of CD8 in facilitating the T cells’ hunt for a rare foreign pMHC ligand and the induction of rapid T-cell response. Thus, our findings provide a new understanding of how MHC clustering influences multivalent interactions of pMHC ligands with CD8 and TCR on live T cells that regulate antigen recognition, kinetics of intracellular signaling, and the selectivity and efficiency of T-cell responses.
Real-time visualization and quantification of neutrophil activation and function using live-cell analysis
Authors: Bevan Nicola , Lovell Gillan , O’Clair Belinda , Kelsey Lauren , Szybut Clare , Campwala Hinnah , Jackson Tim , Holtz Nevine , Endsley Eric , Dale Tim , Trezise Del 
Published: January 2023

Abstract
Polymorphonuclear neutrophils (PMNs) have multiple functions during the resolution of inflammation. At the inflammation site, increases in chemokines induce infiltration of PMNs via CXCR receptor activation, resulting in cell clearance via phagocytosis and NETosis. Here we describe characterization of the activation and function of PMNs using IncuCyte® live-cell analysis.
Changes in cell shape and CD marker expression are known indicators of PMN activation. Freshly isolated PMNs were seeded in 96-well plates, in the presence of FabFlour-488 labeled CD11b antibody for live-cell immunocytochemistry. Phase and fluorescence images were captured every 30 min for 6 h with IncuCyte S3. Cell-by-cell analysis enables individual cell segmentation and yields area, shape (eccentricity) and fluorescent intensity metrics. Subsets of PMNs could be analyzed by classifying on each of these parameters. CXCL8 induced a time- and concentration-dependent increase in eccentricity (EC50 0.63 nM) and concomitant increase in CD11b expression (EC50 0.22 nM). CXCL8-induced activation was attenuated by an anti-CXCL8 antibody. CXCL1 or CCL2 yielded little or no change in either parameter.
As a follow up, we assessed CXCL8-dependent chemotaxis using IncuCyte ClearView Chemotaxis plates, phagocytotic activity with IncuCyte pHrodo® E. coli Bioparticles® and PMA-induced NETosis (IncuCyte Cytotox Green). In all three cases robust, time-dependent signal changes were observed, consistent with known PMN function. We conclude that live-cell analysis is a flexible and powerful method for analyzing neutrophil activity, where morphological, protein and functional parameters can be readily quantified and integrated over time.
An advancement in single cell genomics allows for T cell population analysis at high resolution
Authors: Sukovich David J , Taylor Sarah E B , Pfeiffer Katherine A. , Stubbington Michael J T , Lee Josephine Y , Sapida Jerald , Roidan Daniel P. , Barrio Alvaro Martinez , Walter Dagmar , Brix Liselotte , Jacobsen Kivin , Yeung Bertrand , Zhao Xinfang , Mikkelsen Tarjei S 
Published: January 2023

Abstract
Progress in our understanding of immunology and cancer immunotherapies requires a comprehensive view of immune cell behavior and the interactions of these cells with their environment. Recent technological innovations have facilitated the combination of cell-surface protein, transcriptome, immune repertoire, and antigen specificity measurements from the same single cells, providing thorough and high-throughput lymphocyte characterization.
Using the 10x Genomics Single Cell Immune Profiling Solution with Feature Barcoding technology along with oligo-conjugated antibodies and peptide-MHC (pMHC) Dextramers®, we performed multi-omic characterization of PBMCs from cytomegalovirus (CMV) seronegative and seropositive patients. Next generation sequencing libraries were made following the 10x Genomics workflows, where transcriptome and immune repertoire libraries are generated alongside libraries from DNA barcodes conjugated to antibodies or pMHC.
Full length, paired TCRα/β sequences with specificity to known CMV antigens were identified in the seropositive donor, but not in the seronegative donor. Interestingly, a large Epstein Barr Virus (EBV) pMHC specific T cell expansion was identified in the CMV seronegative donor, suggesting an active EBV response. Moreover, the combination of transcriptomic and cell surface protein information resulted in an increase in resolution of cell type identification. This multi-omic workflow allowed the identification of enriched amino acid motifs within the TCR sequences that contained novel and known CDR3 amino acid sequences specific to CMV.
These technological advancements provide new biological insights that are critical for progress in the field.
Controlled temp for cell plating eliminates room temp cell settling in 96-well plates, reducing Edge Effect and improving cell conditions
Authors: Henn Alicia D. , Darou Shannon , Henn Stassa P.D. , Frank Allayna M. , Alm Kersti , Yerden Randy 
Published: January 2023

Abstract
The “edge effect” has always limited the use of edge wells of widely-used 96-well plates, reduced useable assay space by 37%, increased materials, time, and costs for these assays. Methods reported to reduce edge effect include leaving the plates at room temperature (RT) for extended cell settling times (30–120 min). We previously reported that controlling conditions to 37° C during cell plating prevents thermal currents and non-random distribution of cells seen during cell settling in edge wells. Here we report studies with human A549 lung carcinoma cells, used for in vitro viral assays, and human bone marrow mesenchymal stromal cells (MSC). Our null hypothesis is that there would be no difference in cell density or viability between cells plated/settled at constant 37° C and extended cell settling times at RT conditions. We plated A549 or MSC under controlled conditions in modular cell handling and incubation chambers in the Xvivo System. We pre-incubated plates for 30, 60, 90, and 120 min at RT and compared these with cells handled at constant 37 degrees. We used the HoloMonitor M4 (PHI, Inc) to monitor cell settling and assessed cell density at 24 and 48 hrs later using crystal violet. We found that cell settling at RT resulted in delayed cell attachment, delayed time to first division, and different cell densities as compared with cell handling at 37° C. Cell agitation and thermal variation was still seen between center and edge wells during cell settling with medium warmed to 37 degrees before traditional cell plating at RT. We concluded that full-time control of 37° C during cell plating is necessary to prevent cell density and cell viability edge effect that occurs during routine handling of cells under RT conditions.
The Immune Epitope Database enables and accelerates research
Authors: Salimi Nima , Vita Randi , Mahajan Swapnil , Overton James A , Dhanda Sandeep Kumar , Martini Sheridan , Cantrell Jason R , Wheeler Daniel K , Sette Alessandro , Peters Bjoern 
Published: January 2023

Abstract
The Immune Epitope Database and Analysis Project (IEDB) is a freely available resource funded by the National Institute of Allergy and Infectious Diseases, to advance immunology research. The database allows easy searching of all published experimental data characterizing antibody and T cell epitopes studied in humans, non-human primates, and other animal species. Epitopes involved in infectious disease, allergy, autoimmunity, and transplant are included. The data were derived from more than 19,000 publications. The IEDB also hosts tools that assist in the prediction and analysis of B cell and T cell epitopes. In recent years, the IEDB has updated its search interface and added highly sought after data, such and antibody and TCR sequence information. We will detail how the IEDB can be used to facilitate immunological research.
Intracellular pairing of multiple mRNA species at the single-cell level using DNA Origami
Authors: Ulrich Peaches Rebecca , Schoettle Louie , Dubois Courtney , Jones Kylee , Chaudhary Neelam , Anderson Karen S. , Blattman Joseph 
Published: January 2023

Abstract
Current research has demonstrated the importance of identification of antigen-specific T cells for use in adoptive cell therapy to treat cancer. By identifying rare T cells, we may be able to determine highly effective reagents for tumor regression. A major problem in identifying TCR for clinical applications has been development of high-throughput methods for obtaining CDR3 sequence information from TCRαβ pairs from individual cells. To this end, we have developed novel DNA origami nanostructures with “bowtie”-barcoded capture probes to simultaneously capture human TCRαβ mRNA from single cells. Origami nanostructures are delivered into T cells via electroporation, and TCRα and β mRNA are co-captured onto integral nanostructure probes. Individual TCRαβ sequences are then reverse transcribed and appended to unique origami-specific bowtie oligonucleotide barcodes that can be matched in downstream next-generation sequencing. We have validated this approach using a Jurkat T cell line, expressing known TCRαβ sequences. Upon the synthesis of DNA origami with TCRα and β capture probes, Jurkat T cells were transfected with the origami scaffold, resulting in the capture and amplification of TCRαβ sequences. Validation using Sanger sequencing confirmed the capture of barcoded TCRα and TCRβ CDR3 sequences. Using a custom “bowtie” dual-barcoding system, we have developed an approach for single cell analysis of multiple mRNA species. Although our current focus is on the TCR repertoire, the “bowtie”-barcoded probes may be modified to capture sequence information from virtually any two genes of interest.
Innovative drug discovery for autoimmune diseases aiming at BAFF signaling pathway
Authors: Yoshimoto Keiko , Suzuki Katsuya , Seki Noriyasu , Sugahara Kunio , Chiba Kenji , Takeuchi Tsutomu 
Published: January 2023

Abstract

Background and Purpose
It is well known that BAFF (B cell activating factor) signaling is involved in the pathogenesis of autoimmune diseases. To search for specific inhibitors against BAFF signaling, we have established our original high throughput screening system and discovered a pyrroloprymidine derivative, BIK-13, which inhibits BAFF binding to its receptor, BR3. In this study, we explored the possibility of BIK-13 as a drug to treat autoimmune diseases.

Methods
Human PBMC were stimulated in vitro with a mixture of BAFF, IL-21, and anti-IgM and anti-CD40 antibodies in the presence of BIK-13. Differentiation of B cells and the expression level of Activation-induced cytidine deaminase (AID) in the cells were analyzed by FACS and qPCR, respectively. The amounts of IL-6, IL-10 and IgG in the culture supernatants were measured by ELISA. Serum levels of an anti-dsDNA antibody, IL-6 and IL-10 in BIK-13-treated autoimmune model mice, MRL/lpr, were measured by ELISA. The proportion of B cells among splenocytes and infiltration of lymphocytes into organs of the mice were analyzed by FACS and immunohistochemistry, respectively.

Results
In vitro experiments revealed that BIK-13 suppressed 1) differentiation of B cells, 2) AID expression and 3) IL-6, IL-10 and IgG production in a dose dependent manner. BIK-13 decreased the serum levels of an anti-dsDNA antibody, IL-6 and IL-10 in MRL/lpr mice. Notably, infiltration of B cells into lacrimal glands and the proportion of B cells among splenocytes were remarkably suppressed in BIK-13-treated mice.

Conclusion
Our data collectively suggest that BIK-13, a low molecular weight BAFF-signaling inhibitor, is a promising drug candidate for autoimmune diseases by suppressing B cell activation.
SINGLE-TARGET PAN-LIVER AUTOIMMUNE DISEASE-SUPPRESSING NANOMEDICINES
Authors: Umeshappa Channakeshava Sokke , Singha Santiswarup , Blanco Jesus , Shao Kun , Nanjundappa Roopa Hebbandi , Yamanouchi Jun , Pares Albert , Serra Pau , Yang Yang , Santamaria Pere 
Published: January 2023

Abstract
Peptide-major histocompatibility complex class II (pMHCII)-based nanomedicines displaying tissue-specific autoantigenic epitopes can blunt specific autoimmune conditions by re-programming cognate antigen-experienced CD4+ T-cells into disease-suppressing T-regulatory type 1 cells. Here, we show that single pMHCII-based nanomedicines displaying epitopes from primary biliary cholangitis (PBC)- or autoimmune hepatitis (AIH)- relevant mitochondrial, endoplasmic reticulum or cytoplasmic antigens can broadly blunt PBC, AIH and Primary Sclerosing Cholangitis in various murine models in a non-disease-specific manner without suppressing general or local immunity against infection or metastatic allogeneic tumors. Thus, CD4+ T-cell autoreactivity against liver-enriched autoantigens in liver autoimmune inflammation is not disease-specific and can be harnessed to treat various liver autoimmune diseases broadly.
Angiotensin-converting enzyme inhibitor suppresses Type I interferon responses, decreases neuroinflammation and improves depression-like behavior in MRL/lpr lupusprone mice
Authors: Sriram Uma , Nocito Cassandra , Hand Michelle , Lubinsky Cody , Seliga Alecia , Zuluaga-Ramirez Viviana , Winfield Malika , Persidsky Yuri 
Published: January 2023

Abstract
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multi-organ damage. Interferon- α (IFN-α) is a central mediator in disease pathogenesis. Neuropsychiatric lupus (NPSLE) is one of the most common manifestations of human SLE often causing depression. Administration of IFN-α in patients with chronic viral infections or cancers causes depressive symptoms. Angiotensin-converting enzyme (ACE) is part of the kallikrein-kinin/renin-angiotensin (KKS/RAS) system that regulates many physiological processes, including inflammation and brain functions. It is known that ACE degrades bradykinin into inactive peptides. We have previously shown in an in vitro model of mouse bone-marrow-derived dendritic cells that the effects of captopril (an ACE inhibitor) on IFN responsive gene inhibition were reversed when bradykinin receptors were blocked, suggesting that ACE, in part, acts via kinin receptors to bring about the suppressive effects. We used the MRL/lpr lupus-prone mouse model, an established model to study NPSLE, to check the in vivo effects of captopril on IFN-induced immune responses, neuroinflammation, and behavior. Exposing MRL/lpr mice to IFN-α increased depressive-like behavior and enhanced ACE expression in the brain. Administering captopril in MRL/lpr mice decreased expression of IFN responsive genes in brain and kidney, decreased microglial activation (IBA1 expression) and reduced depressive-like behavior as assessed by the forced swim test. Thus, ACE inhibitors may have potential for therapeutic use for systemic and neurolupus.