bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cognate CD40-CD40L interaction is important for maintenance of thymic B cells. (IRM8P.705)
Authors: Fujihara Chiharu , Williams Joy , Jeon Hyein , Hodes Richard 
Published: January 2023

Abstract
B cells are present in the thymus in numbers similar to dendritic cells and epithelial cells, but the functional roles of thymic B cells and the mechanisms that support this population are unclear. Previously, we showed B cells are essential for endogenous superantigen-mediated thymocyte negative selection. In this study, we investigated mechanisms regulating thymic B cells. Thymic B cell frequency and absolute number are markedly lower in TCRa KO mice, indicating the necessity of SP-T cells for maintenance of thymic B cells. Thymic B cell numbers are also reduced in mice expressing a TCR Tg in absence of antigenic ligand, but dramatically increased in mice expressing both TCR and corresponding ligand, demonstrating TCR recognition and thymocyte activation strongly influence thymic B cells. To elucidate molecular interactions that underlie the requirement for activated T cells, we tested the role of CD40L expressed on activated SP-T cells interacting with CD40 to mediate thymic B cell maintenance. In CD40L and CD40 KO, thymic B cell frequency is substantially reduced. In mixed BM-chimeras in which CD40 KO and WT donor-derived cells co-exist, cell autonomous CD40 expression is important for thymic B cell maintenance, whereas mixed BM-chimeras using MHCII KO and WT showed that cell autonomous MHCII expression plays no detectable role. Thus, CD40L-CD40 cognate interaction, but not MHCII-TCR cognate interaction, between T cells and B cells supports the thymic B cell population.
CRACR2A encodes a novel Ca2-binding Rab GTPase important for T cell receptor signalling (IRM10P.748)
Authors: Gwack Yousang , Srikanth Sonal , Kim Kyun-Do , Gao Yuanyuan , Ghosh Shubhamoy 
Published: January 2023

Abstract
Ca2+ signalling is important for T cell stimulation and its impairment leads to immune deficiency. In T cells, Ca2+ entry is mediated by CRAC (Ca2+-release activated Ca2+) channels that consist of the pore component, Orai1 and the endoplasmic reticulum Ca2+ sensor, STIM1. We recently identified CRACR2A (CRAC channel regulator 2A) as an interacting partner of Orai1 and STIM1. Here we investigate the role of a novel isoform of CRACR2A that is predominantly expressed in immune cells. CRACR2A contains multiple functional domains including Ca2+-binding motifs and a C-terminal Rab GTPase domain. Different from small Rab GTPases, CRACR2A utilized a two-step activation mechanism of GTP binding and T cell receptor (TCR) stimulation to mediate its translocation into the immunological synapse. Defect in GTP binding or hydrolysis activity results in loss and gain of function respectively. CRACR2A played a predominant role in Ca2+ and mitogen-activated protein kinase (MAPK) signalling pathways. Furthermore, CRACR2A-deficient T cells showed impairment in Th1 differentiation. In support of these data, mice with T cell-specific deletion of CRACR2A showed a strong resistance to experimental autoimmune encephalomyelitis. Previously, small GTPase has been shown to play an important role in TCR signaling. Our study identifies the unexpected usage of a large Rab GTPase as a molecular switch to regulate TCR signalling pathways.
Structural mechanism of TCR-pMHC catch bond revealed by molecular dynamics, mutagenesis, and single-molecule kinetics analyses (IRM10P.752)
Authors: Liu Baoyu , Chen Wei , Martinez Ryan , Lou Jizhong , Zhu Cheng , Evavold Brian 
Published: January 2023

Abstract
Mechanical force is increasingly recognized as an important axis in regulation of T cell function. Recently, we showed that force elicits agonist-specific TCR-pMHC catch bond (where force counterintuitively prolongs bond lifetime), which provides a biophysical mechanism of force-triggered T cell Ca2+ flux (Liu et al., Cell, in press). Here, we performed steered molecular dynamics (SMD) simulation and single-bond lifetime experiments to investigate the molecular mechanism of TCR-pMHC catch bond. SMD simulation of 2C TCR-agonist dissociation showed that pulling TCR-pMHC bond leads to characteristic rotation of MHCI α1/α2 domain relative to TCR variable region, yielding multiple new atomic interactions (e.g., hydrogen bond) that are absent in crystal structure. Force-regulated bond lifetime measured on naïve 2C T cells validated two major predictions of such catch bond mechanism. (1) Pulling with a geometry that favors the relative domain rotation (by pulling pMHC β chain rather than c-terminal of heavy chain) produced a more pronounced catch bond; (2) a single point mutation on pMHC that abolishes new hydrogen bond formation eliminated catch bond and lowered EC50 (proliferation) by more than three orders of magnitude with only two fold decrease in affinity and a longer zero force lifetime. These data highlight a dynamic perspective of TCR-pMHC structure that reveals novel insights on TCR-pMHC interaction and T cell function.
Regulation of the intracellular localization of the transcription factor Ets1 (IRM10P.754)
Authors: Dalton Justin , Blair Sarah , Garrett-Sinha Lee Ann 
Published: January 2023

Abstract
Ets1 is a DNA binding transcription factor that regulates B and T cell responses. To activate transcription Ets1 must be present in the nucleus of the cell. The major isoform of Ets1 (designated p54) has both a nuclear localization signal (NLS) and a nuclear export signal (NES). We have identified a novel isoform of Ets1 (p68) having an alternate N-terminus with 71 distinct amino acids that replace the first 28 amino acids of p54. Unexpectedly, we found that transfected p68 is localized in the cytoplasm of cells, rather than the nucleus where p54 is found, despite the fact that p68 contains both the NLS and NES sequences of Ets1. This suggests that sequences in the first 28 amino acids of p54 Ets1 regulate its nuclear localization. Contained within this region of p54 Ets1 is a lysine residue (K15) known to undergo sumoylation. We made point mutations in this site (to either alanine or arginine) and showed that this residue is essential for nuclear localization of Ets1. Studies are currently underway to determine whether sumoylation of Ets1 is required for its nuclear localization. Interestingly, there may be a conserved mechanism that regulates nuclear localization of Ets proteins. This interpretation is based on our observation that another Ets family member Elf5 also requires an intact N-terminus to be localized within the nucleus of the cell. Our studies predict that nuclear localization of Ets proteins may be a regulated event and under the control of signaling pathways.
NAADP mediated calcium release from acidic stores plays a critical role in downstream physiological responses of naïve CD4 and CD8 T cells (IRM10P.757)
Authors: Ali Ramadan , Camick Christina , Wiles Katherine , Slama James , Giovannucci Daivd , Wall Katherine 
Published: January 2023

Abstract
Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ mobilizing second messenger discovered to date and has been implicated in Ca2+ signaling in human Jurkat lymphoma T cells via ryanodine receptor activation. In contrast, the role of NAADP in Ca2+ signaling or the identity of the Ca2+ stores targeted by NAADP in conventional naïve T cells has not been investigated. In the current study, we demonstrated for the first time to our knowledge the importance of NAADP in the generation of Ca2+ signals in murine conventional naïve T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the impact of NAADP on the generation of Ca2+ signals evoked by TCR stimulation and the role of this signal in downstream physiological endpoints such as proliferation, cytokine production, and other responses to stimulation of murine naïve T cells. Moreover, we demonstrated that acidic stores in addition to the endoplasmic reticulum are the relevant Ca2+ stores that were sensitive to NAADP in naïve T cells.
Prevention of enteric disease by peritoneal cells from Bacillus subtilis exopolysaccharide (EPS)-treated mice (MUC4P.840)
Authors: Jones Sara , Knight Katherine 
Published: January 2023

Abstract
Beneficial microbes modulate host immune responses and prevent pathogen-induced inflammation; the molecular mechanisms involved in this process are only recently being elucidated. The enteric pathogen Citrobacter rodentium shares genetic and phenotypic similarities to the human pathogen enteropathogenic E. coli. We previously demonstrated that probiotic B. subtilis, but not an exopolysaccharide (EPS) null mutant, protected mice from C. rodentium-associated disease. We also showed that purified EPS were sufficient to prevent disease when administered i.p. 24 hr prior to pathogen infection. In addition, we found that B. subtilis and EPS failed to protect TLR2 KO and TLR4 KO mice, suggesting that EPS-mediated protection utilizes both of these pathways. Since EPS bind peritoneal macrophages and because EPS are administered i.p., we hypothesized that peritoneal cells may contribute to protection. To test this idea, we adoptively transferred peritoneal cells from EPS-treated mice to naïve mice and found that these cells could prevent disease. We also show that TLR4 and TLR2 are required by peritoneal cells and non-peritoneal cells, respectively. We hypothesize that B. subtilis EPS mediate their protective effects through TLR4-expressing macrophages. Adoptive transfer studies with sorted cells from wt and TLR4 KO mice will be performed to test this idea.
The commensal bacteria metabolites short chain fatty acids positively regulate epithelial innate immune responses in the gut (MUC4P.847)
Authors: Kim Myunghoo , Kang Seung Goo , Park Jeong H. , Yanagisawa Masashi , Kim Chang H. 
Published: January 2023

Abstract
Short chain acids (SCFAs) are highly produced by gut commensal bacteria and play important roles in regulating immune responses. SCFAs can regulate epithelial cells an dimmune cells, and some of the functions are mediated through GPR41 and GPR43. Epithelial cells highly express both GPR41 and GPR43, but the functions of these receptors in regulation of epithelial innate immunity remain unclear. To investigate this, we utilized mice deficient in expression of GPR41 or GPR43. When the gut barrier function was breached with 50% ethanol, GPR41-/- and GPR43-/- mice were defective in expression of inflammatory cytokines (IL-6, IL1b and TNFa) and chemokines (CXCL1 and CXCL2) in response to invading bacteria. When the mice were infected with C. rodentium, the KO mice were delayed in mounting immune responses and clearing the pathogen. On the other hand, wild type mice with increased levels of SCFAs were more efficient in expression of the inflammatory factors. The results of our bone marrow transfer and in vitro culture experiments indicate that epithelial GPR41 and GPR43 play critical roles in mediating the SCFA signal. SCFAs induced a series of signaling events from the surface receptors to ERKs and to ATF2 activation to express the inflammatory factors. These processes are required to recruit and activate immune cells to clear pathogenic bacteria. Our results revealed a novel positive role of short chain fatty acids and their receptors in regulating intestinal immunity.
Vitamin A deficient mice have a decreased ability to clear the enteric pathogen, Citrobacter rodentium and increased mortality during infection (MUC4P.851)
Authors: McDaniel Kaitlin , Restori Katherine , Kennett Mary , Ross A. , Cantorna Margherita 
Published: January 2023

Abstract
Vitamin A is essential for normal immune function and vitamin A supplementation has been shown to decrease childhood mortality from infectious respiratory and diarrheal diseases. In this study, we determined that vitamin A sufficient (A+) mice were able to clear a Citrobacter rodentium infection while vitamin A deficient (A-) mice shed over 10,000 C. rodentium/g feces up to 37 d following infection. A- mice also had increased crypt hyperplasia compared to A- mice indicating that C. rodentium is a chronic infection in A- mice. Supplementation of A- mice with retinoic acid (RA) at d 0 or 14 post-infection resulted in the clearance of C. rodentium. A- mice had decreased frequencies of colonic CD8αα+ Tcrβ+ T cells and an increased frequency of CD4+ Tcrβ+ T cells. A- mice had reduced survival rates (58%) following C. rodentium infection while 100% of A+ mice survived. RA supplementation beginning on the day of infection resulted in 100% survival of the A- mice. The increased lethality of the C. rodentium infection in A- mice corresponded with the systemic spread of C. rodentium. T cells are critical for the prevention of the systemic spread and increased lethality of C. rodentium infection since mice without T cells are unable to clear an infection. The data suggest that vitamin A regulation of mucosal and systemic T cells is critical for survival and clearance of a C. rodentium infection.
The microbiome regulates the homeostasis of host invariant natural killer T (iNKT) cells (MUC4P.830)
Authors: An Dingding , Oh Sungwhan , Olszak Torsten , Neves Joana , Avci Fikri , Erturk-Hasdemir Deniz , Zeissig Sebastian , Blumberg Richard , Kasper Dennis 
Published: January 2023

Abstract
Symbiotic bacteria intimately participate in host physiological processes and impact host health and disease, although details at the molecular level largely remain unknown. We discovered that symbiotic Bacteroides species produce sphingolipids to modify the host antigen environment of the invariant natural killer T (iNKT) cells, a key immune modulator recognizing CD1d-restricted lipids. Using gnotobiotic technology and analytical tools, we found that bacterial glycosphingolipids, specifically one molecular fraction called GSL-Bf717, block iNKT cell activation and inhibit cell proliferation in early life. As a result, the total colonic iNKT cell number is restricted in adulthood and the host is protected when challenged by iNKT cell-mediated oxazolone colitis. These results suggest that intestinal bacterial sphingolipids are critical for establishing colonic iNKT cell homeostasis and host tolerance to environmental challenges. In addition, we find that the regulation depends on exposure to these symbiotic molecules in a critical early-life window, which, if missed, has irreversible impacts on host. This study contributes to our understanding of the essential role microbes play in host health and disease with precious molecular details that are largely lacking. The unique inhibitive power of GSL-Bf717 can be harnessed to provide therapeutic interventions in human autoimmune and allergic disorders where iNKT cell activation is detrimental.
Cadherin-11 regulates pulmonary fibrosis in bleomycin-induced lung injury (MUC5P.865)
Authors: Pedroza Mesias , George Teresa , Agarwal Sandeep 
Published: January 2023

Abstract
Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible pulmonary disorder with unknown effective treatment. IPF is characterized by excessive myofibroblast differentiation in the alveolar airways. Epithelial-to-mesenchymal transition (EMT) is the differentiation of fibroblast-like phenotype from epithelial cells resulting in myofibroblast accumulation. Cadherin-11 (Cad11) expression on hyperplastic alveolar epithelial cells is a novel finding in our lab. Hence, we hypothesized that Cad11 contributes to the development of pulmonary fibrosis through EMT. Our aims consist of determining: 1) The role of Cad-11 in EMT; and 2) The contribution of Cad11 deficiency and neutralization in the intraperitoneal (IP) bleomycin (BLM) model of pulmonary fibrosis. Preliminary findings show that deficiency and neutralization of Cad11 in this model is associated with attenuated pulmonary fibrosis and myofibroblast accumulation. In vitro studies demonstrated that Fc-Cad-11 was able to stimulate MLE-12 cell lines to produce Col1α expression while knockdown of Cad-11 via siRNA in these epithelial cell lines prevented the increase of fibrotic markers. These findings demonstrate that Cad-11 contributes to the development and progression of pulmonary fibrosis. The novelty of this project will be the identification of mechanisms by which Cad11 regulates EMT. This project will support additional translational studies to develop Cad11 as a therapeutic target for pulmonary fibrosis.
Direct isolation of untouched human neutrophils from whole blood in as little as 25 minutes (TECH2P.870)
Authors: Tabatabaei-Zavareh Nooshin , Le Fevre Tim , Kokaji Andy , McQueen Karina , Fairhurst Maureen , Thomas Terry , Eaves Allen 
Published: January 2023

Abstract
Neutrophils are the first line of defense against microbial infections. They sense pathogen-associated molecular patterns through a wide range of receptors including toll-like receptors. Once activated, neutrophils eliminate infection via the phagocytosis of pathogens, releasing proinflammatory cytokines and antimicrobial peptides and producing reactive oxygen species (ROS). The procedures to enrich neutrophils from human whole blood involve at least two steps to remove red blood cells (RBCs) and unwanted leukocytes. For example, neutrophils may be separated from other leukocytes using density gradient centrifugation. However, they are still mixed with RBCs which are then lysed. Alternatively, RBCs are first depleted using lysis or sedimentation followed by fluorescence-activated cell sorting or magnetic cell separation to remove unwanted leukocytes. Here we describe a one-step immunomagnetic, column-free cell separation method which does NOT require RBC lysis, sedimentation or density gradient centrifugation. Non-neutrophils and RBCs are labeled with antibodies and cross-linked to magnetic particles. The labeled cells are removed using an EasySepTM magnet. The complete procedure takes 25-35 minutes. The purity of isolated neutrophils is 96±3% (n=14). The isolated neutrophils are functional and produce ROS upon activation with PMA. This fast isolation method of untouched, quiescent neutrophils facilitates the studies of neutrophil function in inflammatory diseases.
CD25 blockade with daclizumab alters regulatory T cell dynamics in RRMS patients (THER3P.880)
Authors: Huss David , Mehta Devangi , Sharma Akanksha , You Xiaojun , Riester Katherine , Sheridan James , Amaravadi Lakshmi , Elkins Jacob , Fontenot Jason 
Published: January 2023

Abstract
Regulatory T cells (Treg) mediate immune tolerance and depend on IL-2 for homeostasis and function. The biology of IL-2 is pleiotropic and complex. As a growth factor for both regulatory and conventional T cells, IL-2 is able to both potentiate and limit adaptive immune responses. Adding to this complexity, IL-2 can signal through a high-affinity (IL-2Rαβγ) and intermediate-affinity (IL-2Rβγ) receptor. Thus, the response to IL-2 at the single cell level is dependent on both receptor expression and local IL-2 concentration. Treg dysfunction is implicated in the pathogenesis of relapsing-remitting multiple sclerosis (RRMS). Daclizumab, a humanized monoclonal antibody that binds the IL-2 receptor alpha chain and prevents its association with IL-2, has demonstrated efficacy in patients with RRMS, despite causing a reduction in Tregs. These seemingly contradictory observations led us to investigate Treg dynamics in placebo- and daclizumab-treated RRMS patients in the SELECT clinical trial. We report that CD25 blockade reduces Treg numbers, yet Tregs that remain preserve IL-2 signaling capabilities through the intermediate affinity IL-2Rβγ and maintain epigenetic marks characteristic of a phenotypically stable and functional Treg population. Furthermore, Treg dynamics did not associate with treatment efficacy or development of cutaneous adverse events, indicating that the effect of daclizumab on Treg dynamics may be independent of the therapeutic mechanism of action.
Improvement in treatment of experimental colitis in mice by using recombinant Lactococcus lactis with surface-displayed affibody against TNFα (THER4P.889)
Authors: Strukelj Borut , Perse Martina , Ravnikar Matjaz , Lunder Mojca , Cerar Anton , Berlec Ales 
Published: January 2023

Abstract
TNFα is one of the most studied pro-inflammatory cytokines in the pathology of irritative bovel disease (IBD), including Crohn's disease and ulcerative colitis. The current therapy for the treatment of mid-to severe IBD is composed by corticosteroids and parenteral administration of monoclonal antibodies against TNFα with systemic side effects. In order to develop an oral administration of anti-TNFα agent, we have introduced an affibody against TNFα as a binding molecule. A recombinant Lactococcus lactis was developed by means of expression of anti-TNFα affibody as a fusion protein with three functional parts: signal Usp45 protein for the secretion to the microenvironment in gut, TNFα-binding affibody domain and peptidoglycan-bynding AcmA autolysin protein that enables attachment of peptidic construct to the surface of L. lactis. Such recombinant bacteria were characterized by the capability to bind Alexa Fluor 488-conjugated TNFα. By means of flow cytometry we clearly demonstrated a distinct shift in fluorescence between control cells and TNFα-binding cells. Furthermore, mice model with induced experimental colitis was used for animal studies. After oral administration of wild type L. lactis (control group) and recombinant anti-TNF α L. lactis, we observed significantly decrease in the concentration of TNFα in the stool as well as general mucosal improvement in group treated with recombinant anti-TNF lactic acid bacteria.
PIKfyve, a class III lipid kinase, is required for TLR-induced type I IFN production via modulation of ATF3 (THER4P.890)
Authors: Cai Xinming , Xu Yongyao , Kim You-Me , Loureiro Joseph , Huang Qian 
Published: January 2023

Abstract
Type I IFN plays a key role in anti-viral responses. It also has been shown that deregulation of Type I IFN expression following abnormal activation of TLRs contributes to pathogenesis of systemic lupus erythematosus. In this study we find PIKfyve, a class III lipid kinase, is required for endolysosomal TLR-induced expression of type I IFN in mouse and human cells. PIKfyve binds to phosphatidylinositol 3P and synthesizes phosphatidylinositol (3,5)P2, and plays a critical role in endolysosomal trafficking. However, PIKfyve modulates type I IFN production via mechanisms independent of receptor and ligand trafficking in endolysosomes. Instead, pharmacological or genetic inactivation of PIKfyve rapidly induces expression of transcription repressor ATF3, which is necessary and sufficient for suppression of type I IFN expression by binding to its promoter and blocking its transcription. Thus, we have uncovered a novel phosphoinositide-mediated regulatory mechanism that controls TLR-mediated induction of type I IFN, which may provide new therapeutic indication for PIKfyve inhibitor.