bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel function of CCL27 in the localization of skin-homing CCR10+ lymphocytes
Authors: Davila Micha L. , Xiong Na
Published: January 2023

The skin contains an abundance of resident lymphocytes that play vital roles in the first line of defense. Dysregulation of the skin immune system could lead to diseases such as psoriasis and atopic dermatitis; studying the immune cells that contribute to skin immune homeostasis is crucial to understanding these diseases. Our lab has found that various subsets of skin-homing lymphocytes that express the chemokine receptor CCR10 have homeostatic functions. The skin specific ligand to CCR10 is CCL27. We found that the expression pattern of CCL27 in the skin of mice is similar to humans: low expression at a young age with a gradual upregulation into adulthood. This upregulation of CCL27 does not require the stimulation of skin colonizing bacteria, as germ free mice did not significantly differ from specific pathogen free mice in their expression of CCL27, suggesting an intrinsic regulatory mechanism for CCL27 expression. CCR10-expressing cells were found to concentrate near the hair follicles, suggesting a role for CCL27/CCR10 in the localization of these cells to specific microstructures. Preliminary analysis of skin-draining lymph node sections from CCR10+/EGFP vs CCR10EGFP/EGFP mice show differential localization of lymphocytes around the vessel-rich medulla. Further, lymphatic endothelial cells from the skin draining lymph nodes express CCL27. These findings indicate a novel role of CCL27 in directing the migration and localization of CCR10+ cells within the skin-draining lymph nodes and skin.
T Cell-Directed IL-17 production by Lung Granular γδ T cells is Coordinated by a Novel IL-2 and IL-1β Circuit
Authors: Menoret Antoine , Buturla James A. , Xu Maria Mei , Svedova Julia , Kumar Sanjeev , Rathinam Vijay A. , Vella Anthony T.
Published: January 2023

Immune-mediated lung injury is considered the result of an exacerbated innate immune response, although a role for adaptive lymphocytes is emerging. αβ T cells specific for S. aureus enterotoxin A orchestrates a typical innate-like Tγδ17 response during lung injury. However, the mechanism by which a specific T cell response drives an innate immune response in the lung is unclear. Here, we show a role for IL-2 triggering IL-17 production by lung granular γδ T cells as IL-17 synthesis was reduced by IL-2 blocking mAbs in vitro and in vivo. Granular γδ T cells in the lung constitute a rare population, hence Mass Cytometry (CyTOF) was utilized to characterize this population. CyTOF analysis revealed that lung γδ T cells responded directly to IL-2 as evident from STAT5 phosphorylation and RoRγt expression. IL-2 receptor blocking mAbs and JAK inhibition impaired STAT5 phosphorylation and IL-17 release. Moreover, inhalation of S. aureus enterotoxin A induced IL-2 secretion and caspase-1-dependent IL-1β activation to drive IL-17 production. This T cell-mediated inflammasome dependent IL-17 response is maximum when lung Tγδ17 cells were sequentially stimulated first with IL-2 then IL-1β. Further investigation is still needed to identify the cellular source of IL-1β. Interestingly, when IL-2 is given therapeutically to cancer patients it carries a known risk of lung injury that is largely indistinguishable from that seen in sepsis. Hence, this novel mechanism reveals therapeutic targets treating both acute lung injury and high dose IL-2 toxicity in cancer.
Cellular stress mediated by the local microenvironment regulates the homeostatic loss of tissue-resident memory CD8 T cells in different compartments of the lung
Authors: Hayward Sarah L. , Scharer Christopher D. , Takamura Shiki , Kumar Gaurav , Boss Jeremy M. , Kohlmeier Jacob E.
Published: January 2023

Lung-resident memory T cells (lung TRM) are important for protective heterosubtypic immunity against influenza viruses; however, the efficacy of this cellular immunity wanes over time. To better understand the mechanisms leading to the decline of protective cellular immunity, we investigated the long-term maintenance of airway and parenchyma lung TRM. In contrast to circulating flu-specific memory T cells and TRM in other tissues, lung TRM numbers in the airways and parenchyma steadily decline for several months following influenza infection. All flu-specific lung TRM decline at similar rates regardless of expression of the tissue residency markers CD69 and CD103. Parabiosis experiments demonstrate that established lung TRM in the airways or parenchyma do not freely interchange with circulating memory T cells. Thus the gradual loss of these cells is likely due to increased cell death in the tissue. In support of this, airway and parenchyma lung TRM show increased apoptosis compared to systemic flu-specific memory T cells during homeostasis. To identify epigenetic and transcriptional programs specific to lung TRM, we performed parallel RNA-seq and ATAC-seq on flu-specific memory CD8 T cells from the airways, lung parenchyma, and spleen. We found that lung TRM populations have increased expression of genes associated with cellular stress, and airway TRM in particular have a signature associated with amino acid starvation and ER stress. Finally, intratracheal transfer experiments determined that the airway environment was sufficient to program the lung TRM transcriptional signature. Together, these data show that the decline of lung TRM is a direct result of cellular stresses driven by the lung microenvironment.
The natural suppression of respiratory CD8 T cell memory by monocyte-derived dendritic cells (moDC)
Authors: Reagin Katie L. , Slade Chris D. , Klonowski Kimberly D.
Published: January 2023

Heterosubtypic CD8 T cell immunity can protect mice and humans against reinfection with highly mutagenic viruses like influenza. Studies in mice attribute this protection to CD8+ tissue resident memory cells (TRM) residing in the lung and airways. However, both live attenuated influenza vaccines and natural influenza infection fail to protect humans from seasonal influenza. Interestingly, unlike TRM derived from other mucosal infections, the majority of TRM in the lung parenchyma are devoid of CD103 and CD69 expression, suggesting bona-fide TRM are either not generated or stably maintained. Using a mouse model, we show respiratory infection evokes the selective migration of CD11c+CD11b+CD103+CCR2+PD-L1hi moDCs to the lung-draining lymph nodes in response to a variety of pathogens (influenza, RSV, VSV) 72–96 hours post infection, coordinate with T cell priming. These moDC develop in the context of systemic and intranasally VSV challenged animals and influenza infected mice, but only emigrate from the bone marrow after respiratory infection. Lymph node immigration is coordinate with regional CCL2 expression. Importantly, the selective depletion of moDCs using CCR2-DTR bone marrow chimeras numerically enhances CD103+CD69+ TRM formation and increases local IFN-ɣ and TNF-α after heterologous virus challenge. In summary, our studies show that moDCs selectively suppress TRM formation after respiratory infection. Ongoing studies seek to understand the molecular mechanism(s) used by moDCs to alter CD8 T cell programming and TRM development.
Comparison of two platforms for the measurement of fg/mL concentrations of protein biomarkers using single molecule arrays and digital ELISA: the benchtop reader Quanterix SR-X™, and the fully automated analyzer HD-1™
Authors: Lambert Jeremy , Vonk Ariel , Mendes Muriel , Song Linan , Svancara Danielle , Baig Shazia , Rissin David , Nie Shuai , Hanlon David , Patel Purvish , Duffy David , Shan Dandan , Chang Lei , Johnson Joe
Published: January 2023

Digital ELISA (Enzyme Linked Immunosorbent Assay) based on single molecule arrays (Simoa) has improved sensitivity of traditional ELISA from picomolar (10−12M) to femtomolar (10−15M), increasing the quality and quantity of biomarkers that can be measured for health and disease. Digital ELISA counts signal generated from single immunocomplexes formed on superparamagnetic beads confined in arrays of femtoliter-sized wells in which fluorescent molecules are highly concentrated.

Quanterix has developed digital ELISA assays in a fully-automated instrument (Simoa HD-1 Analyzer), ideal for use in pharmaceutical companies, drug discovery, clinical research and other areas necessitating full automation and high throughput. Recent advancements in the Simoa technology and workflow have been integrated into the new SR-X benchtop reader, with a smaller footprint and more flexible workflow. Operators prepare assays in microtiter plates at the bench in a semi-automated format similar to traditional ELISA, with the notable exception that plates are preserved by drying after assay completion, and can be read immediately or the next day.

We present results comparing performance of the following Simoa assays on SR-X to HD-1: PSA; HIV p24; Tau; Neurofilament-light; PD-L1; TNFa; IL-10; IL-17A; IL-6 and a neurology multiplex panel consisting of NF-L, tau, GFAP and UCH-L1. Measured sample levels correlated with R2 values from 0.96 to 1.00, with average LOD and LLOQ within 1.4 and 1.5 fold of HD-1, respectively. Inter-assay precision ranged from 4.0 to 11.2% CV across assays. Operators tested full-plates from start to finish within 1 – 2 hours (1/2 hour hands on time) and a read time of 2 hours (5 minutes hands on time).
Functional Interrogation and Mining of Natively-Paired Human VH:VL Antibody Repertoires
Authors: DeKosky Brandon J. , Wang Bo , Timm Morgan , Lee Jiwon , Normandin Erica , Misasi John , Kong Rui , McDaniel Jonathan R. , Delidakis George , Leigh Kendra E. , Niezold Thomas , Ploquin Aurélie , Viox Elise G. , Fahad Ahmed , Cagigi Alberto , Leung Kwanyee , Yang Eun Sung , Kong Wing-Pui , Voss William , Schmidt Aaron G. , Moody M. Anthony , Ambrozak David , Henry Amy R. , Laboune Farida , Ledgerwood Julie E. , Graham Barney S. , Connors Mark , Douek Daniel C. , Sullivan Nancy , Ellington Andrew D. , Georgiou George
Published: January 2023

Next-Generation sequencing has become an essential tool in the analysis of antibody responses in the settings of health, vaccination, and disease. However, immune receptors comprise two chains encoded by separate mRNA strands, and conventional NextGen sequencing fails to identify the native pairings encoded by individual lymphocytes. To overcome this limitation we have applied recent technical advances in high-throughput sequencing and functional analysis of complete antibodies (i.e., paired heavy and light chain sequencing) to generate a comprehensive understanding of the antibody response to vaccination and natural infection. Here we present a new technology to screen natively-paired human antibody repertoires from millions of B cells. Libraries of natively-paired variable region heavy and light (VH:VL) amplicons were expressed in a yeast display platform that was optimized for human Fab surface expression, and the resulting libraries were interrogated for binding to viral vaccine antigens via FACS paired with next generation sequencing. Using our method we identified HIV-1 broadly neutralizing antibodies (bNAbs) from an HIV-1 slow progressor and high-affinity neutralizing antibodies responding to an Ebola virus glycoprotein vaccination. These next-generation approaches are providing detailed molecular feedback on immune receptor responses and are informing the design and discovery of new vaccines and therapeutics.
A high-resolution, genome-scale promoter interactome in human T follicular helper cells implicates novel causal genes at SLE GWAS loci
Authors: Wells Andrew D. , Johnson Matthew , Manduchi Elisabetta , Le Coz Carole , Leonard Michelle , Lu Sumei , Hodge Kenyaita , Romberg Neil , Chesi Alessandra , Grant Struan
Published: January 2023

Genome-Wide Association Studies (GWAS) have implicated >60 loci in the susceptibility to systemic lupus erythematosus (SLE). However, GWAS reports signals in non-coding genomic regions, not the precise location of culprit genes. Chromatin conformation capture (3C) technologies that detect physical contacts between regions of the genome offer a powerful opportunity to map disease variants to target genes. We developed a massively parallel, high-resolution method to characterize the genome-wide interactomes of 36,691 promoters of protein-coding, noncoding, antisense, snRNA, miRNA, snoRNA and lincRNA genes in any cell type. Using this method, we generated promoter interactomes of primary human T follicular helper (TFH) cells from healthy tonsil, a cell type relevant to SLE as TFH operate upstream of pathogenic autoantibody-producing B cells. These sub-1kb TFH promoter interactome datasets were intersected with maps of TFH open chromatin generated by ATAC-seq and SLE SNPs from the 63 candidate loci, resulting in detection of consistent interactions between genes and accessible SNPs at 48 loci. We find that ~25% of accessible SLE SNPs interact with the nearest gene, e.g. STAT4 and IKZF3, while ~75% of accessible SNPs ‘skip’ the nearest gene to interact with distant genes, e.g. LCLAT1 at the ‘LBH’ locus, and the master TFH transcription factor BCL6 at the ‘LPP-TPRG1’ locus. Gene ontology analysis confirms that genes directly implicated by SNP interactions reside in SLE-relevant networks while ‘nearest to SNP’ genes do not. In conclusion, high-resolution, 3-dimensional promoter interactions with accessible, disease-associated SNPs in disease-relevant tissue connect variants to relevant genes with high apparent accuracy.
Robust prediction of clinical outcomes using cytometry data
Authors: Hu Zicheng , Glicksberg Benjamin , Butte Atul
Published: January 2023

Flow cytometry and mass cytometry are used to diagnose diseases and to predict clinical outcomes. Here, we propose a novel strategy to predict clinical outcomes using cytometry data without cell gating (CytoDx; accessible at github.com/hzc363/CytoDx). CytoDx is able to predict the response to influenza vaccine using heterogeneous datasets, demonstrating that it is not only accurate but also robust to batch effects and cytometry platforms.
Sensitive detection of human drug-induced liver injury by measurement of microRNA-122 using single molecule arrays
Authors: Lambert Jeremy , Rissin David , Duffy David
Published: January 2023

Drug-induced liver injury (DILI) is a major challenge in clinical medicine and drug development. MicroRNA-122 (miR-122) is a sensitive and specific biomarker for hepatocyte injury that has received formal regulatory support. A major roadblock to its widespread adoption is a lack of a fit-for-purpose, scalable assay that can provide quantification of circulating miR-122 with appropriate accuracy and time-to-result. We have developed a PCR-free, sensitive and highly specific assay for measuring miR-122. The assay is based on the hybridization of miR-122 to a peptide nucleic acid (PNA) probe attached to superparamagnetic beads, followed by the specific incorporation of a biotinylated nucleobase. The biotin labels are then labeled with an enzyme, and single enzymes are detected using single molecule arrays (Simoa).

The miR-122 single molecule assay had a limit of detection (LOD) of 500 fM, approximately 500 times more sensitive than a corresponding analog bead-based assay. The specificity of the assay to a single base mismatch in the microRNA sequence was >107-fold ((highest concentration of mismatched target with signal below LOD)/(LOD)). As measured by our single molecule assay, all patients with DILI had higher concentrations of miR-122 in their serum compared to healthy controls, and the concentrations of miR-122 measured correlated closely with those determined using the gold standard PCR (r2 = 0.93; slope = 0.64).

This novel single molecule assay enabled the accurate measurement of miR-122 in the serum of patients with DILI. This assay could have utility in clinical practice as it has the potential to deliver the user-independent sensitivity and time-to-result that are needed to inform pre-clinical and clinical decision making.
Dynamic live cell imaging of immune cell interactions using microfluidics cell trap
Authors: Park Jun , Chen Cindy , Yeh Victor , Booth Ross
Published: January 2023

While providing indispensable insight into immune processes, the live cell imaging of immune cells poses unique challenges due to their non-adherent nature. Suspension cells such as immune cells can easily move away from the FOV (field of view) during imaging by any physical perturbations such as introduction of agonist or even simple media change, often necessitating the use of surface coating to artificially immobilize cells. In addition, unlike adherent cells, interacting suspension cells form aggregates, making it very difficult to track and analyze individual cells during imaging. Previously, we have described the novel microfluidic-based dynamic live cell imaging platform CellASIC ONIX2, where microenvironmental parameters such as the perfusion of media/reagent, temperature, and gas compositions can be precisely controlled on demand via software during the entire imaging experiment. We now report further utility of the system with a new non-adherent cell trap culture plate comprising an array of walled ~100μm square pads with a 12μm height restriction and 3μm wall gap, allowing physical trapping of immune cells within FOV while enabling their natural movement, and preventing vertical cell stacking of aggregated cells while preserving cell-cell interactions. Using Jurkat T cells and super antigen loaded Raji B cells, we demonstrate dynamic live cell imaging of immune cell interactions. Unlike systems where cells are artificially immobilized, we show that these cells are extremely motile and behave in much more complex and dynamic fashion during their interactions. The technology should benefit live cell imaging of any suspension cells and is amendable for wider applications in addition to immune cell imaging.
Ultrasensitive Quantification of Inflammatory Biomarkers in Breast Cancer Survivors
Authors: Osier Nicole D. , Slote Claire , McGreevy Emily , Henneghan Ashley
Published: January 2023

Background

Inflammatory changes in breast cancer survivors, have been linked to symptoms including sleep disturbance and fatigue. While inflammation is well-studied, limitations in detection of inflammatory proteins in human serum may obscure scientific understanding; traditional methodologies (e.g. enzyme-linked immunosorbent assay (ELISA)) often cannot detect beyond the picomolar level).

Purpose

This study describes new methodologies for evaluating biomarkers by comparing IL-6- levels in the serum of breast cancer survivors previously obtained in the parent study using the BioPlex200 System (Luminex®) to those obtained in this study using the SMCxPRO (MilliporeSigma).

Methods

The principles outlined in the Declaration of Helsinki and U.S. Government regulations (45 CFR Part 46; 56 FR 28003) were followed. Following Institutional Review Board (IRB) approval and informed consent, survivors of treated Stage I–III breast cancer (6 to 10 years prior) were recruited into the parent study. Serum and self-reported measurements of sleep and fatigue (e.g. Pittsburgh Sleep Quality Index, Epworth Sleepiness Scale, Patient-Reported Outcomes Measurement Information System Fatigue scale) were collected at a single time point. A bead-based assay system was then used to perform single molecule counting and these results were compared to those previously obtained using conventional methods.

Results

Inflammatory changes are detectable using both systems, but the SMCxPRO assay has a lower limit of quantification. Femtomolar-level sensitivity may result in identification of meaningful associations that could not be determined otherwise. Future research efforts to replicate and expand upon these findings are needed.
REAlease™ Immunomagnetic Separation Technology with reversible labeling for positive selection of leukocytes
Authors: Evaristo Cesar , Steinbrück Philipp , Pankratz Jennifer , Yu Zhong , Dose Christian
Published: January 2023

Immunomagnetic enrichment of leukocytes is an important technique in both research and clinical applications. Accordingly, there is a continuous need for improved, reliable cell separation methods. Current enrichment strategies using magnetic labeling of the target cells allow for highly efficient isolation, but in some instances removal of residual cell surface labeling after isolation is of high interest. To this end, we have combined the benefits of positive selection by MACS® Technology, the proven state-of-the-art method for the isolation of functional, viable cells, with a novel technology enabling the removal of both superparamagnetic beads and antibody fragments. REAlease™ Technology provides an easy and fast solution for the highly specific isolation of unlabeled leukocytes directly from PBMCs. We present our latest results on cell separation with REAlease Technology based on key human immune cell markers, i.e., CD3, CD4, CD8, CD19, and CD56. The benefits of these releasable labels are demonstrated for the isolation of important subsets such as CD3−CD56+ NK cells, and CD4+ and CD8+ cell populations from a single sample. Using various assays, we show that functionality and viability of the target cells are preserved without cell activation.
Isolation of functionally unaffected cells Straight From buffy coat, LRSC or Leukopak
Authors: Winkels Gregor , Schlemminger Maike , Weber-Lohmann Silke , Kolnik Martin , Yu Zhong , Höher-Peters Susanne
Published: January 2023

Enrichment of leukocytes from blood products like buffy coat, leukocyte reduction chambers (LRSC) or leukapheresis material can be time consuming and could influence the physiology of the target cells.

Our new StraightFrom cell separation reagents were specifically developed to minimize time and handling steps, omitting the need to perform density gradient centrifugation methods or erythrocyte lysis prior to isolation thereby preserving cell intergrity.

Based on positive isolation, Th cells (CD4), cytotoxic T cells (CD8), Monocytes (CD14), B cells (CD19) or NK cells (CD56) can be isolated from complete buffy coats, LRSC or half pack LPs within less than 30 minutes with purities above 95%. The positive enrichment process enables thereby the consecutive enrichment of more than one cell population from a sample and is automatable.

Using various assays, we show that functionality (e.g activation and expansion, cytokine secretion, ROS production) and viability of the target cells are preserved without cell activation. Ultimately, this optimized magnetic cell separation technology allows the isolation of physiologically relevant cells in a quick and easy manner.
Development of a murine model resembling human lupus in mice parasitized with Trichinella spiralis
Authors: Nevárez-Lechuga Christian Irene , Sánchez-Barbosa Sandra , Landa-Saldívar Carla , Wong Carlos , Wong-Baeza Carlos , Escobar-Gutiérrez Alejandro , de-la-Rosa-Arana Jorge-Luis , Baeza Isabel
Published: January 2023

Deregulation of Th1 and Th17 immune responses is fundamental in the development of autoimmune diseases, like Systemic Lupus Erythematosus that is a chronic inflammatory autoimmune disease characterized by the production of autoantibodies that cause inflammation and damage to different organs. New therapeutic approaches for autoimmune diseases have been based on the fact that nematodes such as Trichinella spiralis decrease the inflammatory response by inducing the production of anti-inflammatory cytokines, which are potent modulators of T cell function. Recent studies have shown that the proteins secreted by T. spiralis, or the infection itself, induce a polarized Th2/Treg response and the production of regulatory cytokines, which are associated with the amelioration of autoimmune diseases such as colitis, respiratory tract inflammation, type 1 diabetes and experimental autoimmune encephalomyelitis. In this work, mice were infected and, after the life cycle was completed, a procedure to develop lupus was performed. The level of autoantibodies and antibodies anti-Trichinella were detected as well as the levels of Th1 and Th2 cytokines. Here, we show the relevance of studying the immune response in a murine model resembling the human lupus in the presence of T. spiralis muscular larvae, because it provides new information on the immunoregulation of lupus and reveals the effect on its progression.
Single-walled carbon nanotubes target neutrophils and Ly-6Chi monocytes and localize to joints in murine models of arthritis
Authors: Hung Shu-Chen , Rajasekaran Narendiran , Zhu Shoujun , Ma Zhuoran , Ghosn Eliver , Mellins Elizabeth D.
Published: January 2023

Rheumatoid arthritis (RA) is a chronic autoimmune disease that often leads to joint damage. The etiology of RA remains unclear, but new insights and therapeutic strategies have been facilitated by murine models. In the K/BxN serum transfer (STA) model, arthritis is triggered by autoantibodies against glucose-6-phosphate isomerase. The SKG model has a ZAP70 point mutation that reduces TCR signaling. After zymosan exposure, SKG mice develop arthritis, mediated by autoreactive Th17 cells. Joint inflammation in these models shares many features with RA, including leukocyte invasion, synovitis, and bone resorption. Neutrophils and Ly-6Chi monocytes are known to play critical roles in these models.1–4 Here, we investigated single-walled carbon nanotubes (SWNT) as candidate drug carriers to specifically target pathogenic cells in arthritic joints. Using FACS analysis, we found that, 2h and 4h after IV administration of SWNT, nearly 80% Ly-6Chi monocytes in blood had taken up SWNT in STA and SKG model, respectively. Near-infrared (NIR) photoluminescence whole-animal imaging of the STA model showed that SWNT were first detected in arthritic joints 3h post injection (p.i.) and reached a maximal level 24h p.i., In contrast, in non-arthritic mice, SWNT detected in joints remained low 24h p.i., suggesting SWNT can specifically target cells homing to or present in arthritic joints. In SKG mice, 2d p.i., SWNT were found in Ly-6Chi monocyte and in neutrophils in bone marrow and liver, but not in blood and spleen. FACS analysis indicated specific uptake of SWNT labelled with Cy5.5 in monocytes and neutrophils in human blood. We conclude that SWNT is a potential carrier to target drugs to pathogenic neutrophils and monocytes in arthritic joints.
T cells directed against different myelin antigen epitopes cooperate to induce autoimmune encephalomyelitis in mice
Authors: Rojano Elba Mirelle , Robinson Rachel R. , Forsthuber Thomas
Published: January 2023

Multiple Sclerosis (MS) is the most common demyelinating autoimmune disease of the central nervous system (CNS). Important insights have emerged into the pathogenic mechanisms that drive the inflammatory events and demyelination in MS, however, much less is known about the events initiating this disease. The murine model of MS, experimental autoimmune encephalomyelitis (EAE), is usually induced through injection with a high dose of single immunodominant peptides found on the myelin sheath of the CNS. Low doses of single peptide do not induce EAE or result in very low disease incidence. In individual MS patients, autoimmune T cell-reactivity is usually directed against multiple myelin antigen epitopes and fluctuates significantly over the course of disease. Thus, we hypothesize that MS is triggered by the emergence of autoreactive T cells directed against multiple myelin epitopes, which together synergize to induce the disease. To test this concept in an animal model of EAE, we explored the effect of induction of EAE using combinations of low dose peptides akin to a “multiple hit” mechanism implicated in other disease conditions. We found that EAE can be induced through the immunization of mice with a combination of low dose myelin peptides from different myelin proteins, which individually are suboptimal to induce disease. The disease observed after multi-peptide immunization is comparable or more severe than with optimal doses of peptide traditionally used to induce EAE. This research provides new insights into the potential mechanisms underlying the induction of MS and could lead to novel approaches for immunotherapy.
Immune Modulation and Vascular Repair Sustain Pancreatic Beta Cell Regeneration To Reverse Late Stage Type 1 Diabetes
Authors: Ukah Tobechukwu Kenneth , Cattin-Roy Alexis N. , Miller Mindy M. , Zaghouani Habib
Published: January 2023

Type 1 diabetes (T1D) is a chronic inflammatory disease which manifests when the pancreatic β-cells are destroyed by cells of the immune system. We have previously shown that antigen-specific therapy with Ig-GAD2, an Ig chimera carrying glutamic acid decarboxylase 206–220 sequence (GAD2), induces regeneration of β-cells and reverses T1D when intervention is made at the pre-diabetic stage. However, Ig-GAD2 could not reverse T1D when the treatment is made at the onset of disease (diabetic stage). Surprisingly, endothelial precursor cells (EPCs), alongside Ig-GAD2, sustain reversal of the disease at the diabetic stage. This combination therapy proved effective in driving immune modulation and repair of the islet vasculature leading to formation of new endogenous β-cells that were able to thrive and restore long-lasting normoglycemia. We now have evidence that combination therapy leads to formation of healthy islets by inducing division of residual β-cells and differentiation of precursor cells. Furthermore, while the pancreas is cleared of immune infiltration during recovery from disease, both the lymph nodes and spleen displayed a significant reduction in Th17 cells and the disease did not rebound. These findings suggest that combination therapy is able to drive long lasting immune modulation and islet vascular repair which provides a micro-environment suitable for regeneration of β-cells at late stages of the disease. These circumstances are relevant to humans as intervention could be made at early as well as late stages after diagnosis.
Contribution of Protein Arginine Methyltransferase (PRMT)5 to House Dust Mite Asthma
Authors: Guerau-De-Arellano Mireia , Sengupta Shouvonik , Amici Stephanie , Webb Lindsay , Weiss Kelly , Asosingh Kewal
Published: January 2023

Severe asthma affects 2.4 million Americans and 23.8 million people worldwide and accounts for the majority of total asthma hospital costs. Most severe asthma is mediated by Th17/neutrophilic responses, alone or combined with Th2 and/or Th1 responses. In contrast, Treg responses are deficient. Protein Arginine Methyl Transferase (PRMT) 5 catalyzes symmetric dimethylation (SDM) of arginine on histones and other proteins, thereby regulating gene expression. PRMT5 promotes murine Th1/Th17 responses in central nervous system autoimmunity. However, its impact on asthma has not been established. We evaluated the role of PRMT5 on mouse models of house dust mite (HDM) asthma. PRMT5 and its symmetric demethylation activity were increased in lung infiltrating cells during Th2/Th17-mediated HDM severe asthma, suggesting a role for PRMT5 in asthma pathogenesis. To determine the contribution of PRMT5 to HDM asthma pathogenesis, we used first-in-class PRMT5-specific inhibitors developed at The Ohio State University. PRMT5 inhibitor HLCL65 modulated Th1, Th2 and Th17 differentiation and suppressed airway hyperreactivity in Th2/Th17 models of house dust mite (HDM)-induced asthma. Overall, these results support a role for PRMT5 in severe asthma pathogenesis.
Rifampicin ameliorates inflammatory responses in asthma experimental animal model
Authors: Kang Tae Jin , Park Jun Woo , Prakash Annamneedi Venkata , Boo Kyung-Jun , Kim Yu Ri , Seong Ju-won
Published: January 2023

Drug repositioning is the application of known drugs and compounds to treat new diseases and is the process of identifying new uses for drugs outside the scope of their original medical indication. Our study previously revealed anti-atopic dermatitis activity of rifampicin, which is used for the treatment of tuberculosis. We herein investigated the effect of rifampicin on asthma. Asthma was induced in BALB/C mice and the mice were treated with rifampicin by oral administration. The inflammatory responses were alleviated in the rifampicin-treated group. Serum The level of inflammatory cytokines was ameliorated in bronchoalveolar lavage fluid (BALF) from mice treated with rifampicin. We next examined whether rifampicin has anti-atopic activity via suppression of mast cell activation. Rifampicin suppressed the release of β-hexosaminidase and histamine from human mast cell (HMC)-1 stimulated with compound 48/80. Treatment with rifampicin inhibited secretion of TNF-α and PGD2 in mast cells activated by compound 48/80. The mRNA expression of COX-2 was reduced in the cells treated with rifampicin. Our findings suggest that rifampicin has efficacy and could be applicable to develop potent therapeutic reagent for the treatment of asthma.
Clonal expansion within circulating plasmblast populations lends support for an infectious disease etiology of Kawasaki disease
Authors: Hicar Mark , Hoffman Jonathon , Sojar Hakimuddin , Baron Sarah
Published: January 2023

Kawasaki Disease (KD) is a childhood vasculitis, marked by prolonged fevers and coronary artery inflammation/aneurysms in near one quarter of those untreated. The cause remains unknown; however, epidemiology and demographic data support a single preceding infectious agent may lead to KD.

Plasmablasts (PBs) are a stage of transitional B-cells that lead to plasma cells, the long-lived antibody producing cells of the bone marrow. After initial infection, peripherally circulating PB populations are enriched for cells with antibodies against the preceding infection. We have recently published data showing children with KD have similar PB responses to children with infections. We sought to define the antibody characteristics, including clonality, of these PBs during KD.

We used antibody repertoire next-generation sequencing to characterize memory and PB populations. Additionally, pairing of heavy and light chains was performed with Chromium Single Cell Gene Expression (10x Genomics, Pleasanton, CA) using the Human B cell Single Cell V(D)J Enrichment Kit.

From subject 24, antibody sequences using VH4-34 and a 19 amino acid length complementarity determining region 3 showed a massive expansion between day 4 and 6 of fever. Chromium single cell sequencing produced over 946 heavy and light chain paired sequences. Sequence comparison showed 40% of sequences demonstrated markers of clonal expansion, which represented 100 clonal groups. One clonal group (24-01) reflected the massive clonal expansion (VH4-34, CDR3 19) previously shown.

Conclusions

This clonal expansion within plasmablast populations supports that Kawasaki disease is caused by an infection. Antigen targeting of these monoclonal antibodies is currently being explored.
CD4 T cell transcriptomics reveal novel diagnostic and mechanistic immune signatures of tuberculosis
Authors: Burel Julie G , Lindestam Arlehamn Cecilia S , Pomaznoy Mikhail , Seumois Gregory , Greenbaum Jason A , Vidanagama Dhammika , Gunasena Bandu , Tippalagama Rashmi , de Silva Aruna D , Taplitz Randy , Vijayanand Pandurangan , Sette Alessandro , Peters Bjoern
Published: January 2023

As part of the Human Immune Project Consortium (HIPC) program, we are aiming to decipher CD4 T cell immune signatures of tuberculosis (TB) in the context of controlled (latent TB) and uncontrolled (active TB) infection. We found commonalities and differences in TB-specific CD4 T cell immune responses of latent TB vs active TB subjects. Both cohorts had higher frequency of peptide-reactive CD4 T cells compared to TB uninfected individuals, and showed strong upregulation of type I and type II interferon genes in response to antigen-specific in vitro stimulation. Conversely, we also detected differences in the transcriptome of CD4 T cells between latent vs active TB subjects that can inform us on TB-specific T cell immune signatures of controlled infection versus disease. In particular, active TB was associated with higher expression of genes associated with inflammation (TNF, IL-1 and TLR signaling) whereas latent TB was enriched for genes associated with metabolism, oxidative phosphorylation and neutrophil-mediated immunity. Looking at inter-individual variability, we also identified that some latent TB infected subjects showed similar expression patterns for a subset of genes to those with active TB, suggesting this molecular pattern might contain mechanistic and prognostic information on the risk of developing active TB. Overall, our approach has identified a plethora of TB-associated T cell immune signatures that allowed us to gain mechanistic insights into the key CD4 T cell components contributing to TB protective immunity. Additionally, our findings have the potential to be translated into new diagnostic and prognostic tools, in particular, identifying latent TB infected individuals at risk of developing active TB.
Eosinophilic esophagitis has a sub-epithelial regulatory adaptive immune response
Authors: Lin Edwin , Lucendo Alfredo , Appelman Henry , Delker Don , Hazel Mark , Wechsler Michael , Yandell Mark , Clayton Frederic
Published: January 2023

Introduction

Eosinophilic esophagitis (EoE) is food-triggered immune esophageal disease characterized by dysphagia and food impaction. The pathophysiology of EoE is poorly understood. Prior eosinophilic esophagitis studies showed abundant IgG4 with IgG4+ plasma cells in esophageal subepithelial tissue.

Methods

Seven esophagectomies and one deep biopsy from patients with active EoE were compared. Seven uninvolved esophageal areas from surgical resections for tumor and autopsy were used as controls. Histology, immunostains, RNA in situ hybridization, and RNA-seq findings were compared with EoE epithelial biopsy RNA-seq.

Results

The superficial lamina propria (LP) of EoE patients has dense fibrosis, tertiary lymphoid tissue with abundant B cells, IgG4 plasma cells, and Foxp3+ regulatory T cells. RNA-seq confirms this, with abundant B cell related transcripts, IGHG4, organized lymphoid tissue marker CCL21, and regulatory T cell markers. IL-10 in situ hybridization is present in mononuclear cells, in and near lymphoid aggregate germinal centers. Edema and degranulating mast cells are absent. By RNAseq, the LP of both eosinophilic esophagitis patients and controls have abundant thrombospondin-1, which could activate TGFβ. Nuclear immunostaining for phosphoSMAD2 and phosphoSMAD3 (suggesting active TGFβ), is present the epithelium and the superficial lamina propria.

Conclusion

The EoE LP immune response has abundant IgG4 plasma cells, regulatory T cells, and a predominantly immunoregulatory transcriptome. Some LP immunoregulatory features are present even in controls. Further work is needed to determine whether the immunoregulatory infiltrate contributes to the dysphagia-inducing subepithelial fibrosis.
Harnessing single cell analysis to characterize the role of MAIT cells in ulcerative colitis
Authors: Sharma Siddhartha M , Gioia Louis , Abe Brian , Holt Marie , Costanzo Anne , Kain Lisa , Su Andrew , Teyton Luc
Published: January 2023

Ulcerative colitis (UC) is an increasingly prevalent form of inflammatory bowel disease, characterized by chronic T cell mediated inflammation of colonic mucosa. UC is likely caused by a confluence of immune dysregulation, genetics, gut microbiome dysbiosis, and environmental factors. Mucosal associated invariant T (MAIT) cells are a sub-population of CD8+ T cells that recognize microbial derived vitamin B2 based antigens and are a significant population in homeostatic mucosa. MAITs are likely a heterogenous population and a subset of MAITs may “link” microbiome dysbiosis, environmental factors and chronic colonic inflammation. To assess the role of MAIT cells in UC, we have analyzed MAITs isolated from matched colonic biopsies, both uninflamed and inflamed mucosa, and blood from a cohort of UC patients. Utilizing MR1 tetramers and single cell analysis techniques, we confirmed a decrease in MAIT cell frequency in peripheral blood of UC patients relative to healthy donors. We have also found an increase in MAIT frequency in inflamed colonic mucosa of UC patients relative to uninflamed mucosa. Single cell gene expression profiling revealed tissue specific differences between blood MAIT cells and colonic MAIT cells as well as a trend for higher expression of activation and pro-inflammatory genes in MAITs isolated from inflamed mucosa. Most intriguingly, single cell TCR sequencing revealed the expansion of a subset of MAITs in inflamed colonic mucosa with a distinct TCRVb and CDR3b sequence. This suggests that a sub-population of MAIT cells in inflamed colonic mucosa are reacting to unique microbial ligands. A new cohort will extend these findings, but our preliminary results support a role for MAIT cells in the pathology of UC.
Serum Antibody Profiling Healthy Toddlers Reveals Low, Intermediate and High Response Cohorts
Authors: van Oers Nicolai S , Pichilingue-Reto Patricia , Raj Prithvi , Dozmorov Igor , Li Quan_Zhen , Wakeland Edward K , Kelly Nancy , de la Morena M. Teresa Teresa
Published: January 2023

During their first two years of life, infant/toddlers produce their own antibodies in response to diverse environmental exposures, infections, and vaccinations. The specificity of these antibodies towards self-antigens, infections, and vaccine components and how they vary among individuals is poorly characterized. We developed a new antigen array comprising autoantigens, infectious agents, and vaccine antigens to assess the serum antibody specificities among a cohort of >150 health toddlers. An analysis of their responses reveals a stratification of healthy toddlers into 3 groups, low, intermediate, and high responders. Among those 16% that are high responders, their serum IgGs were able to bind a diverse array of self-antigens as well as infectious agents. Longitudinal follow-up suggests this pattern is relatively stable over time. Comparing clinical data reveals a significant correlation with the high IgG responder cohort and a family history of asthma and maternal gestational diabetes. Targeted DNA sequencing in the high responder group revealed a strong genetic association signal at the HLA locus, with polymorphisms at this locus associated with high ANA and IgG titers to many antigens. Findings from this study may provide insights into the natural history of human autoantibody formation.
B cell-intrinsic and lymphocyte extrinsic miR-21 drive optimal germinal center and antibody responses
Authors: Schell Stephanie L , Choi Nicholas , Rahman Ziaur S.M.
Published: January 2023

Germinal centers (GCs) generate high affinity B cell responses that are protective against infection but can produce autoreactive responses when dysregulated. MicroRNAs (miRNAs) have been implicated in the regulation of GC fate, though the role of many individual miRNAs remains uncharacterized. Here, we demonstrate that within the GC, miR-21 promotes T follicular helper cell (Tfh), T follicular regulatory cell (Tfr), and GC B cell responses, as well as downstream antibody production. Regulation of the GC response by miR-21 requires multiple mechanisms, occurring in both B cells and innate cells. B cell-intrinsic miR-21 drives GC B cell expansion by promoting B cell activation and proliferation. MiR-21 target gene expression arrays have revealed several putative target genes in B cells, suggesting that miR-21 targets negative regulators of proliferative pathways to enhance B cell proliferation and GC responses. In contrast, Tfh and Tfr are driven by miR-21 in a B and T cell extrinsic manner, likely by innate cells. Our data suggest that miR-21 modifies the cytokine microenvironment, which impacts regulatory T cell expansion and Tfh programs. Therefore, the synergy of B cell intrinsic and lymphocyte extrinsic miR-21 activity is required to generate optimal GC and Ab responses. Of clinical relevance, we also found that upregulation of miR-21 in autoimmune-prone mice drives autoantibody responses, implicating miR-21 in both anti-pathogen responses and its dysregulation in autoimmunity. Therefore, dampening miR-21 function may provide a strategy for autoimmune therapeutics. Alternatively, boosting miR-21 activity in healthy individuals could allow more efficient generation of protective antibody following vaccination.
Regulation of B cell-mediated immune responses by a PX domain-containing protein
Authors: Jung Da-Jung , You Gihoon , Park Areum , Kim You-Me
Published: January 2023

The humoral immunity is mediated by antibodies produced by plasma cells, which are terminally differentiated effector cells of the B cell lineage. The antibody production should be tightly regulated to prevent unwanted responses against self-molecules and efficiently defend the host against foreign antigens. In this study, we aimed to examine the roles of PX domain-containing kinase in B cell-mediated immune responses. PX domain-containing kinase is a cytosolic protein composed of a PX domain, a pseudokinase domain, and a WH2 domain. It is expected that PX domain-containing kinase regulates intracellular trafficking of membrane proteins based on its structural features and previously reported functions. PX domain-containing kinase is most highly expressed in B cells both in humans and mice. Furthermore, several reports indicated that SNPs of the PX domain-containing kinase gene are linked to autoimmune diseases commonly characterized by excessive production of autoantibodies, such as systemic lupus erythematosus, rheumatoid arthritis, and systemic sclerosis. However, the in vivo function of PX domain-containing kinase in immune responses has not been reported. In order to investigate the immunoregulatory functions of PX domain-containing kinase, we firstly generated PX domain-containing kinase-deficient mice. Although PX domain-containing kinase is not essential for the development of immune cells, we found that foreign antigen-induced antibody responses were affected by deletion of PX domain-containing kinase in mice. Currently, we are in the process of uncovering the underlying mechanisms. This study is expected to elucidate the functions of PXK as a novel modulator of antibody-mediated immune responses.
Cell-specific inhibition of SMAD2/3 restores lymph node cellularity and germinal center function in aged mice responding to acute chikungunya virus (CHIKV) infection
Authors: Uhrlaub Jennifer , Yang Yu-Sang Sabrina , Melo Mariane B , Irvine Darrell J , Nikolich-Zugich Janko
Published: January 2023

Targeted delivery of small-molecule inhibitors increases drug efficacy by focusing its action upon specific cell-types without global disruption of signaling pathways. TGFβ is a pleotropic cytokine involved in many cellular processes, including specific regulatory functions during anti-viral immune responses. Previously, we have shown that in aged mice, over-production of TGFβ correlates with decreased immune function. TGFβ neutralization during acute chikungunya virus (CHIKV) infection decreases acute- and chronic- disease severity and improves neutralizing antibody titers. Herein, we selectively interrupted TGFβ signaling during acute CHIKV infection via the delivery of lymph node (LN)-targeting nanoparticles coupled to a small-molecule inhibitor of SMAD2/3 phosphorylation. We show that LN-targeted inhibition of SMAD phosphorylation during the initiation of a viral immune response can restore – to the levels measured in adult mice - the cellular content of the draining lymph node. We further demonstrate that inhibition of TGFβ signaling decreases LN fibrosis and improves the germinal center reaction in aged mice.