bluuebunny/crossref_metadata_2025_split huggingface dataset:
A simple method for assessment of human anti-Neu5Gc antibodies applied to Kawasaki disease (P3263)
Authors: Padler-Karavani Vered , Tremoulet Adriana , Yu Hai , Chen Xi , Burns Jane , Varki Ajit 
Published: January 2023

Abstract
N-Glycolylneuraminic acid (Neu5Gc) is a dietary immunogenic sugar that metabolically incorporates into diverse glycoconjugates in humans. Anti-Neu5Gc antibodies are detected in all human sera, play a role in several inflammation-mediated pathologies and are potential diagnostics of such diseases. Currently, it is difficult to determine their levels in individual human samples due to diverse Neu5Gc-epitopes recognition. Here we describe a simple method for detection of human anti-Neu5Gc antibodies. We exploit the difference between two mouse models that differ only by Neu5Gc-presence (wild-type) or Neu5Gc-absence (Cmah-/- knockout). We characterize mouse serum from both strains by HPLC, lectin and mass-spectrometry. We then use Cmah-/- knockout sera to inhibit all non-Neu5Gc-reactivity followed by binding to wild-type sera to detect overall anti-Neu5Gc response in a single assay. We applied this methodology to characterize anti-Neu5Gc IgG and IgA in sera of Kawasaki disease (KD) patients at various stages compared to controls. KD is an acute childhood febrile disease characterized by inflammation of coronary arteries that untreated may lead to coronary artery aneurysms, thrombosis and myocardial infarction. This estimated response is comparable to the average of detailed anti-Neu5Gc IgG profile analyzed by a sialoglycan microarray. Both assays revealed an elevated response in acute KD patients with normal coronaries compared to patients with aneurysm or dilated coronaries.
Real-time impedance monitoring of pathogen induced apoptosis as a screening method for antiviral drug discovery (P3279)
Authors: Campbell Carmen , Motzfeldt-Laane Carl , Moses Ashlee 
Published: January 2023

Abstract
Many intracellular bacteria and viruses cause host cell death by apoptosis. Antimicrobial agents that block host cell apoptosis are lead drug candidates. To facilitate this, one requires a real-time, automated detection method that monitors pathogen-induced apoptosis to screen for anti-apoptotic drugs. In the current work, we demonstrate that Electric Cell-Substrate Impedance Sensing is a powerful tool for real-time monitoring of pathogen induced cell death in a cell culture system. The impedance biosensor is sensitive enough to detect micro-motion of single cells on an electrode and measures changes in impedance over time. As cells adhere to and spread out across the electrodes, the impedance of the electrode increases relative to the amount of electrode area covered with adhered cells. As virus replicate inside the cells and induce apoptosis, the cell membranes begin to disintegrate and detach from the surface effectively restoring the flow of current resulting in decreased impedance. The method has proven to be remarkably accurate for measuring viral induced cell death in a time and dose dependent manner in both fish and human cell models. Results of the experiments and future applications of this method for antiviral drug discovery will be presented. Reference: Campbell, C.E., Motzfeldt-Laane, M., Haugarvoll, E., and Giaever, I., "Monitoring Viral Induced Cell Death using Electric Cell-Substrate Impedance Sensing." Biosensors and Bioelectronics: 23(4): 536-542 (2007).
Systematically defining murine immunity at the phenotypic and functional levels via mass cytometry (P3283)
Authors: Spitzer Matthew , Hotson Andrew , Bendall Sean , Engleman Edgar , Nolan Garry 
Published: January 2023

Abstract
Despite decades of immunology research in the mouse, no systematic and comprehensive characterization of murine immunity at the proteomic level has been reported. While flow cytometry has been utilized to interrogate immune cells under many circumstances, overlap in fluorochrome emission spectra limits the number of simultaneous parameters available, impeding a thorough analysis of all immune cells and their behavior in complex tissues. Mass cytometry, which couples flow cytometry to time-of-flight mass spectrometry, circumvents this limitation. Here we report the application of single-cell mass cytometry to characterize wild-type murine immunity in three common mouse strains. Using a 40-dimensional panel of immunophenotypic markers, we have assessed the cellular frequency and phenotype of immune cells in the peripheral blood, spleen, lymph nodes, bone marrow, and thymus in an unsupervised manner. Moreover, by combining phenotypic analysis with phospho-specific flow cytometry, we have determined the cell signaling response of immune cells in these tissues to over 20 different stimuli. By characterizing 10 mice per strain, we have defined the ranges of intra-strain and inter-strain variability in immune cell phenotype and behavior. Thus, we report a wild-type murine immune reference map as a resource for the field. We believe this tool will greatly facilitate thorough characterization of pathology, genetic mutations and therapeutic interventions in mouse models.
A novel haploid genetics approach to dissect immune pathways (P3235)
Authors: Shen Jingshi , Dinarello Charles 
Published: January 2023

Abstract
Unbiased loss-of-function genetic screens in model organisms have been major driving forces for modern biological research. However, many biological functions of humans such as adaptive immunity are not found in model organisms amenable to classic genetic screens. Despite tremendous efforts, human somatic cell genetics remains unattainable due to the inability to efficiently induce homozygous mutations in diploid human cells. Our group developed a novel haploid genetics approach to dissect immune pathways in human cells. We take advantage of human myeloid cells lines (e.g. KBM7) that exhibit a haploid karyotype. Retroviral insertional mutagenesis of the haploid cells generates a library of mutant cells with individual genes completely disrupted. First, we validated the haploid mutant library by screening for mutants resistant to the cytotoxicity of diphtheria toxin. After selection, the retroviral insertion sites of the resistant clones were mapped by deep sequencing. We identified HBEGF, the known cell surface receptor for diphtheria toxin. We also isolated multiple endocytic membrane proteins, which likely mediate the internalization of diphtheria toxin. Next, we demonstrated that the haploid mutant library is also suitable for studying complex immune pathways such as the elimination of tumor cells by cytotoxic T lymphocytes. This haploid genetics approach provides a novel and powerful platform for dissecting immune pathways on a genome-wide scale.
Repertoire-wide single-cell linked immunoglobulin DNA libraries (P3247)
Authors: Johnson David , Conant Carolyn , Withey Gary , Löhr Andrea , Hsu Andro , Meyer Everett 
Published: January 2023

Abstract
Deep sequencing is used to broadly characterize mammalian B cell repertoires. DNA from these libraries might be used to produce monoclonal antibodies in vitro, but current methods do not amplify paired heavy and light chain immunoglobulin (Ig). We have developed a method that combines microfluidics and DNA amplification to produce repertoire-wide single-cell linked Ig DNA libraries. We encapsulate hundreds of thousands of single B cells into aqueous-in-oil picoliter droplets. Inside droplets, we perform overlap extension reverse transcriptase PCR, which reverse transcribes and amplifies heavy and light chain Ig separately in early cycles. In later cycles, a linker DNA sequence drives synthesis of a fusion product containing both heavy and light chain Ig. The fusion products encode fragments of monoclonal antibodies (mAbs). We spiked immortal B cell lymphocytes into peripheral blood at several concentrations. Previously, we sequenced the immunoglobulin kappa (IgK) and heavy (IgH) chain variable regions of each cell line. Then, we produced linked Ig repertoire DNA libraries using a set of primers targeting the full variable regions (CDR1, CDR2, and CDR3) for the full IgK and IgH repertoires. We were able to recover expected IgK and IgH pairings for each of the spike-ins, with &lt;5% background. Affinity-selected mAbs from linked repertoire libraries could eventually be used as therapeutics in autoimmunity or cancer, and may have higher efficacy than artificially derived mAbs.
Plasticity in the differentiation of IL-22 producing cells (P6265)
Authors: Shen Wei , Li Wenqing , Feigenbaum Lionel , Hixon Julie , Durum Scott 
Published: January 2023

Abstract
T helper type 22 (Th22) cells are a subset of CD4+ effector T cell that primarily produces interleukin 22 (IL-22), IL-13, and TNF-alpha and have an indispensable role in host defense, barrier function, tissue repair and inflammation. However, the stability and plasticity of Th22 cells have not been elucidated. In this study we developed an IL-22-tdTomato reporter mouse to examine the plasticity of Th22 cells in vitro and in vivo. Briefly, a bacterial artificial chromosome (BAC) was modified to introduce the fluorescent reporter gene into the Il-22 locus. By homologous recombination, the synthesis of the signal peptide of Il-22 in the BAC was disrupted and the tdTomato gene with poly A was inserted immediately after the ATG start site of Il-22, replacing exon 1. Mice were generated that harbor the BAC construct. We found that in vitro generated Th22 cells still maintained their differentiation program in vitro in the early stage of development and could be reprogrammed into Th17 and Th1 cell lineages, but not Th2 cells, in the later stage. To observe Th22 plasticity in vivo, we sorted IL-22 producing cells from mouse T cell transferred colitis model and injected them back to the Rag-/- mouse. It demonstrated that Th22 cells slowly lost their IL-22 expression and converted into Th17 cells in a TGF-beta dependent manner. These results, although suggesting the flexibility of Th22 cells, also indicate active maintenance of their program in vivo.
Dose-dependent suppression of cytokine production from T cells by a novel phosphoinositide 3-kinase delta inhibitor (P6275)
Authors: Way Emily , Chen Kong , Puri Kamal , Kolls Jay 
Published: January 2023

Abstract
The use of specific cytokine inhibitors to treat inflammatory conditions is common but typically utilizes monoclonal antibodies, fusion proteins, and other molecules whose large size necessitates parenteral administration. There remains a significant need for development of effective small molecule cytokine inhibitors for inflammatory conditions. Phosphoinositide 3-kinase (PI3K) plays a critical role in multiple cell signaling pathways, cell cycle progression, and cell growth, and is thus a potential target for drug development. We examined the effect of a novel PI3Kδ inhibitor on CD3/CD28 stimulated T-cell cytokine production. When added to culture after allowing naïve T-cells to polarize into effector T-cells, we observed dose-dependent suppression of multiple cytokines by Luminex and ELISA, including IL-17, IFNγ, and IL-4 from Th17, Th1, and Th2 cells, respectively. Real time PCR confirmed the suppression of cytokine gene expression suggesting the inhibition is mediated at the transcriptional level. However, we do not observe suppressed gene expression of Tbet, GATA3, or RORc, the critical transcription factors for production of those cytokines. A potential mechanism is that the PI3Kδ inhibitor alters nuclear localization of these transcription factors post-translationally. These experiments show effectiveness of this small molecule inhibitor of PI3Kδ activity to modulate inflammation in vitro and exhibit promise as a treatment for in vivo models of inflammation.
Activation of Complement5a receptor on macrophages inhibits Type I Interferon mediated suppression of Th17 cells in lupus (P6277)
Authors: Biswas Partha , Pawaria Sudesh , Maers Kelly 
Published: January 2023

Abstract
Systemic lupus erythematosus is a type I Interferon (type I IFN) driven systemic autoimmune disorder with exaggerated B and T-helper (Th) cells response. Th17 cells, a recently identified T-helper cell known for its ability to produce inflammatory cytokine Interleukin (IL)-17 have been implicated in the pathogenesis of lupus. Since, type I IFN can suppress the generation Th17 cells in an IL-27 dependent manner, it is unclear how pathogenic Th17 cells are generated in the presence of type I IFN in lupus prone mice and patients. Here we show that, activation of Complement5a-receptor (C5aR) on lupus prone macrophages blocked type I IFN mediated IL-27 production and facilitated the development of Th17 cells. C5aR activation on macrophages inhibited IRF1 mediated upregulation of IL-27 gene expression. Consequently, in a pristane-induced lupus model, C5aR-deficient mice had fewer Th17 cells and reduced disease severity in comparison to wild type control mice. Finally, we are able to demonstrate that C5aR activation on macrophages from lupus patients produced diminished level of IL-27 in response to type I IFN stimulation. Collectively, these data clearly indicate an essential role for innate immune signals in the generation of pathogenic Th17 response in lupus. In the long run knowledge gained from this study will be beneficial in designing potential therapeutic strategies to treat lupus patients.
PD-1 interaction with PD-L1 but not PD-L2 on B cells mediates protective effects of estrogen against EAE (P5176)
Authors: Bodhankar Sheetal , Offner Halina 
Published: January 2023

Abstract
Increased remissions in multiple sclerosis (MS) during late pregnancy may result from high levels of sex steroids such as estrogen (E2) and estriol (E3). In fact, treatment with relatively low doses of E2 can protect against clinical and histological signs of MOG-35-55 induced experimental autoimmune encephalomyelitis (EAE) through mechanisms involving PD-1, PD-L1 and B cells. The current study sought to determine more specifically the contribution of PD-L1 and PD-L2 on B cells in E2-mediated protection. Thus, WT, PD-L1 knockout (KO) and PD-L2 KO mice were implanted with E2 pellets prior to EAE induction and the disease course was followed. Unlike PD-L2 KO mice that were fully protected against EAE after E2 treatment, E2-implanted PD-L1 KO mice were fully susceptible to EAE, with increased numbers of proliferating Th1/Th17 cells in the periphery and severe cellular infiltration and active demyelination in the CNS. Moreover, transfer of B cells from MOG-immunized PD-L1 KO or PD-L2 KO donors into E2-preconditioned B cell deficient μMT-/- recipient mice revealed significantly reduced E2-mediated protection against EAE in recipients of PD-L1 KO B cells, but complete protection in recipients of PD-L2 KO B cells. We conclude that PD-1 interaction with PD-L1 but not PD-L2 on B cells is crucial for E2-mediated protection in EAE and that strategies that enhance PD-1/PD-L1 interactions might potentiate E2 treatment effects in EAE and MS.
IL21 is a critical regulator of chronic/persistent auto-immune and allo-immune responses (P5162)
Authors: Khattar Mithun , Schroder Paul , Baum Caitlin , Deng Ronghai , Chen Wenhao , Stepkowski Stanislaw 
Published: January 2023

Abstract
IL21 is critical for T1D development in NOD mice, but its role in sustaining T cell responses against islets during T1D progression remains unexplored. Our studies show that diabetic NOD mice have high levels of serum IL21. We thus hypothesized that IL21 produced by activated CD4 T cells in spontaneously diabetic NOD mice sustains T cell responses to islets. Indeed, IL21 is required for maintaining the function of auto-reactive immune cells during T1D progression as transient therapy with IL21 receptor fusion protein (IL21RFc) was effective in reversing the onset of T1D in NOD mice (n=11) and inducing long-term T1D remission for up to 20 weeks in at least 3 mice. Pancreatic infiltrates of IL21RFc-treated NOD mice contain mostly CD4 T cells that lost surface ICOS expression while containing fewer CD8 T cells, suggesting that IL21RFc inhibited CD4 T cell help to CD8 T cells. In a parallel study, we have shown that IL21 deficient mice were protected from chronic allograft vasculopathy in a MHC class-II mismatched cardiac allograft model. However, IL21 deficient mice acutely rejected fully mismatched cardiac allografts similar to wild type. Thus, we have demonstrated a novel and critical role for IL21 signaling in driving chronic immune responses against auto and allo-antigens. Based on our findings, IL21 has the potential to act as a serum biomarker for early T1D detection and a therapeutic target to abrogate persistent T cell responses in T1D and chronic allograft rejection.
Evaluation of SOCS1 mimetic and antagonist peptides as therapeutic agents (P5152)
Authors: Bedoya Simone , Young Ashley , Wilson Tenisha , Johnson Howard , Larkin III Joseph 
Published: January 2023

Abstract
The intracellular protein Suppressors of Cytokine Signaling1 (SOCS1) plays a critical role in moderating inflammatory cytokine signaling, thereby limiting immune responsiveness. We have shown that a peptide with the kinase inhibitory region of SOCS1 (SOCS1-KIR) inhibits Experimental Autoimmune Encephalomyelitis and rescues SOCS1-deficient mice from perinatal lethality. In contrast, a SOCS1antagonist pJAK2 enhances antiviral immunity to poxviruses. Therefore it is likely that peptides that modulate SOCS1 signaling possess the potential to modulate immune responses, and restore immune homeostasis. To evaluate the translational potential of SOCS1 mimetic peptides, we have assessed their pharmacodynamics by fluorescently labeling the peptides followed by injection into C57BL/6 mice. After 2 hours mice receiving Alexa 647 fluorochrome-conjugated SOCS1-KIR or pJAK2 were sacrificed, and subsequently whole organs and tissue histology were examined for the presence of the labeled peptides. We observed that pJAK2 and SOCS1-KIR were present in the brain, liver, lymph nodes, spleen, kidneys, lungs, and peritoneal cells. Notably, pJAK2 was selectively present within the thymus and heart. On a cellular level, SOCS1-KIR and pJAK2 were differentially present within CD4+ T cells, CD8+ T cells, CD11b+ macrophages, and B cells. These results help to lay the groundwork for the translational use of peptides that modulate SOCS signaling to regulate and/or maintain immune system homeostasis.
Genome-wide DNA methylation study suggests epigenetic accessibility and transcriptional poising of interferon-regulated genes in naïve CD4+ T cells from lupus patients (P4539)
Authors: Sawalha Amr , Jeffries Matlock , Altorok Nezam , Dozmorov Mikhail , Koelsch Kristi , Wren Jonathan , Merrill Joan , McCune W. , Coit Patrick 
Published: January 2023

Abstract
Abnormal T cell DNA methylation and type-1 interferon play an important role in the pathogenesis of systemic lupus erythematosus. We performed a genome-wide DNA methylation study in two independent sets of lupus patients and matched healthy controls to characterize the DNA methylome in naïve CD4+ T cells in lupus. DNA methylation was quantified for over 485,000 methylation sites across the genome, and differentially methylated sites between lupus patients and controls were identified and then independently replicated. Gene expression analysis was also performed from the same cells to investigate the relationship between the DNA methylation changes observed and mRNA expression levels. We identified and replicated 86 differentially methylated CG sites between patients and controls in 47 genes. Importantly, we observed significant hypomethylation in interferon-regulated genes in naïve T cells from lupus patients, suggesting epigenetic transcriptional accessibility of these genetic loci. The hypomethylation in interferon-regulated genes was not related to lupus disease activity. Gene expression analysis showed overexpression of these genes in total but not naïve CD4+ T cells from lupus patients. Our data suggest epigenetic “poising” of interferon-regulated genes in lupus naïve CD4+ T cells, argue for a novel pathogenic implication for abnormal T cell DNA methylation in lupus, and suggest a mechanism for type-1 interferon hyper-responsiveness in lupus T cells.
CD8+ T cells in a normal host limit autoimmune attack by CD-8+ effector T cells (P4541)
Authors: Girtman Megan , Pavelko Kevin , Pease Larry 
Published: January 2023

Abstract
We have developed a mouse model of diabetes by adoptively transferring small numbers of OT1 cells into animals expressing OVA under the rat insulin promoter (RIP-OVA). These cells are activated in vivo with a Picornavirus (TMEV) expressing OVA antigen. Titrating the dose of adoptively transferred cells, changes disease outcome. Mice receiving low amounts of cells develop hyperglycemia, and recover homeostasis. Investigation of recovered animals revealed normal blood glucose regulation. Pancreatic histology of recovered mice showed extensive pancreatic destruction with no or very few remaining insulin-positive islets. While adoptively transferred OT1 cells were present, pancreas infiltrating T cells were largely endogenous. To evaluate the functional importance of the host T cells, we tested disease progression using RAG KO, CD4 KO, and CD8 KO RIP-OVA hosts. Without RAG, CD4, or CD8 we observed increased disease severity. This change is not attributed to homeostatic proliferation in the CD4 and CD8 KO animals, as adoptively transferred OT1 cells behaved similarly in the KO hosts in comparison to RIP-OVA mice. However there is significant homeostatic proliferation occurring in the RIP-OVA RAG KO animals. Our studies reveal the importance of CD4 and CD8 T cells in limiting autoimmune attack in the pancreas. Although populations of CD4 T regulatory cells are well known, the importance of CD8 T cells in limiting inflammation are less well characterized.
The nuclear receptor PPARγ regulates T cell tolerance and germinal center formation via generation of follicular helper T cells (P4545)
Authors: Choi Je-Min , Park Hong-Jai , Choi Jin-Young , Senejani Alireza , Tobiasova Zuzana , Kim Do-Hyun , Bothwell Alfred 
Published: January 2023

Abstract
Peroxisome proliferator-activated receptor gamma (PPARγ) acts as a transcription factor to regulate lipid and glucose metabolism. An emerging role of PPARγ has been suggested that it has important regulatory functions in inflammation and adaptive immunity. However, the intrinsic role of PPARγ in T cells is not fully understood. Here we used CD4-PPARγKO mice to understand the function of PPARγ in T cells. PPARγ deficient T cells produced higher levels of cytokines including IFN-γ, IL-17, or IL-2, and proliferated more than wild type T cells following TcR stimulation with increased phosphorylation of ERK and AKT. Interestingly, PPARγ deficient T cells were prone to differentiate into various helper T cell subsets such as Th1, Th2, Th17, and Th9 cells. Furthermore, aged mice with PPARγ deficient T cells showed increased numbers of follicular helper T cells (TFH cells) and spontaneously develop a moderate autoimmune phenotype with kidney inflammation, enhanced autoantibody production and increased numbers of germinal centers. The abrogation of T cell tolerance was further confirmed with the diminished the expression of IkBα, Sirt1, and Foxo1 to inhibit NF-κB. These results suggest that PPARγ is crucial for maintaining T cell tolerance and germinal center formation and TFH generation to prevent autoimmunity.