bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multiplexing analysis of cell proliferation and cellular functions using a new multicolor panel of fluorescent cell proliferation dyes (P1290)
Authors: Liao Jinfang , Zhao Qin , Wu Yibo , Diwu Zhenjun 
Published: January 2023

Abstract
Flow cytometry combined with fluorescent staining is a powerful tool for monitoring viable cells. CFSE is the most popular fluorescent dye for cell proliferation studies. However, the identical Ex/Em spectra to GFP or FITC make CFSE impossible to use for GFP cells or the applications where a FITC-labeled antibody is used. The other disadvantages of CFSE include the high background fluorescence and severe fluorescence intensity decrease on the 2nd generation of cells. We report the functional analysis of cell proliferation using a new class of multicolor CytoTell™ dyes. CytoTell™ dyes are functionally similar to CFSE since they contain both an esterase-cleavable and a cell-retaining group. They are well excited at major laser lines like 405, 488 or 633nm with multicolor emissions. We demonstrated that Cytotell™ dyes have a few significant advantages over CFSE. 1) They have minimal cytotoxicity and well retained in cells since they do not indiscriminately react with proteins, and have minimal MDR interaction; 2) They exhibit much faster response and are more convenient to use than CFSE. There is no fluorescence intensity gap between 1st and 2nd generation of cells. 3) There is no need to remove medium since CytoTell™ dyes do not react with medium components; 4) They are much more stable than CFSE. The stock solutions can be stored at RT for a few days; and (5). CytoTell dyes can be readily used for the multicolor applications with either GFP cells or FITC-labeled antibodies.
The role of Drak2 in T cell expansion and survival (P1298)
Authors: Harris Tarsha , Creasy Blaine , McGargill Maureen 
Published: January 2023

Abstract
Drak2 is a serine-threonine kinase expressed most abundantly in T cells and B cells. We previously demonstrated that mice deficient in Drak2 (Drak2-/-) are resistant to disease in models of type 1 diabetes (T1D) and multiple sclerosis (MS) due to a defect in the accumulation of autoreactive T cells. Yet, Drak2-/- mice retain the ability to launch an effective immune response to pathogen infections. Current treatments for autoimmune diseases function by suppressing the immune system, which increases susceptibility to infections and tumors. Thus, Drak2 is a great potential therapeutic target to treat autoimmune disease, because its inhibition may alleviate autoimmune disease without compromising immunity to infections and tumors. Despite its potential as a target, it is unclear how Drak2 regulates the accumulation of autoreactive T cells. Our preliminary data show a decrease in the survival of Drak2-/- T cells compared to wildtype T cells following in vitro stimulation that is rescued by co-culture with wildtype T cells, suggesting a role for Drak2 in production of a critical survival factor. In addition, Drak2-/- T cells display defects in cell cycle progression, morphology, and actin polymerization following activation, implicating a role for Drak2 in the regulation of cytoskeletal dynamics. These data emphasize novel roles for Drak2 in T cell accumulation and suggest that, in the absence of Drak2, T cells exhibit defects in both survival and proliferation.
Bacterial polysaccharide exposure limits asthmatic inflammation. (P6051)
Authors: Johnson Jenny , Jones Mark , Cobb Brian 
Published: January 2023

Abstract
The prevalence of allergic diseases, such as asthma, has significantly increased in Western society over the past 30 years. One explanation for this trend is embodied by the hygiene hypothesis, which suggests that decreased microbial exposure may lead to an increased risk of allergic disease. Consistent with this notion, colonization with the common commensal bacteria Bacteroides fragilis or treatment with its purified capsular polysaccharide (PSA) can induce a regulatory population capable of suppressing effector T cell function in vitro and in vivo, including EAE and IBD disease models. Our previous findings revealed a dramatic increase in CD4+CD25+CD45Rblow cells upon stimulation with PSA, and these cells were able to suppress T cell activation in an IL-10 dependent fashion. Here we show that oral treatment of C57Bl/6 mice with PSA generates a FoxP3- population of cells within the spleen, and that these cells are capable of suppressing asthmatic lung inflammation. This activity was given to naïve recipients by the adoptive transfer of CD4+ splenocytes from PSA-treated mice. In contrast to previous data, transferred T cells from IL-10-deficient mice were also able to protect naïve recipients from asthma, ultimately revealing a novel mechanism by which commensal antigen-induced memory T cells influence the peripheral immune system to decrease susceptibility to allergic inflammation.
The microRNA-17 modulates Foxp3+ regulatory T cells activity by targeting Eos (P1073)
Authors: Yang Huang-Yu , Pardoll Drew M , Pan Fan 
Published: January 2023

Abstract
Regulatory T (Treg) cells play a critical role in maintaining self-tolerance and controlling the magnitude of physiologic immune response. We were interested to determine whether Treg differentiation and/or suppressive activity is regulated by miRNA. miRNA are short (~22 nt) noncoding RNAs, expressed from endogenous genes, and are the products of sequential processing of primary RNA polymerase II transcripts mediated by two RNase III enzymes, Drosha and Dicer. The importance of miRNAs in Treg function is clearly demonstrated by studies using Treg specific knockout mice for Dicer and Drosha. Treg cell specific deletion of either Dicer or Drosha caused autoimmune disease indistinguishable from Foxp3 mutation or knockout mice, even though Foxp3 expression level was not changed in these Treg cells. The inflammatory cytokine IL-6 had been found to dampen Treg function, but how IL-6 suppresses Treg function is not understood. Here we report that IL-6 actively affects Eos expression through miR-17, a key component of the miR-17-92 cluster, thus downmodulating Treg suppression function and providing Treg with partial effector activity via de-repression of cytokine genes. Using miR-17-92 knockout mice, we demonstrated that miR-17-92 knockout protected mice from experimental autoimmune encephalomyelitis due to enhancement of Treg suppression but not Foxp3 levels. Our findings support that in the presence of IL-6, Treg function was regulated by miR-17 via targeting Eos.
Neuropilin-1 immune regulation is mediated by the mTOR pathway (P1099)
Authors: Bynoe Margaret 
Published: January 2023

Abstract
We have recently shown that Neuropilin-1 (Nrp1), a cell surface molecule originally identified for its role in neuronal development and angiogenesis, is involved in several aspects of immune function. Our recent study provided the first evidence for a central role of Nrp1 in the regulation of CD4 T cell immune responses in experimental autoimmune encephalitis (EAE), a neuroinflammatory disorder. Using a tissue specific deletion system, we observed that the lack of Nrp1 in CD4+ T cells results in increased EAE severity. These conditional knockout mice exhibit preferential TH-17 lineage commitment and decreased regulatory T (Treg) cell functionality. Conversely, CD4+ T cells expressing Nrp1 suppress effector T cell proliferation and cytokine production both in vivo and in vitro, independent of Treg cells. In this study, we demonstrate that treatment with rapamycin, an immunosuppressive agent that also causes the expansion of Treg cells and inhibits Th17 cell development, exacerbated EAE in Nrp1-/- mice but had a delayed onset in WT mice. Furthermore, rapamycin treated Nrp1-/- but not WT mice produced more IL-17 and IFN-gamma which are known to be pathogenic in EAE. These findings suggest that Nrp1 may mediate its immunesuppressive effects via the mammalian target of rapamycin (mTOR) signaling.
2B4-mediated homotypic interactions among activating NK cells enhances activation and proliferation of NK cells via clustering and polarization of IL-2 receptors (P1101)
Authors: Doh Junsang , Kim Miju , Kim Hye Mi , Lee Kyung-Mi 
Published: January 2023

Abstract
Clustering of activating lymphocytes has been frequently observed during early stage of immune responses, but the importance of such multi-cellular clusters has not been systematically investigated due to the lack of tools. To address it, we fabricated cell-laden microwells that allowed us to precisely control multi-cellular interactionss among activating lymphocytes. The role of contact-mediated homotypic interactions between natural killer (NK) cells during IL-2 activation was examined using the cell-laden microwells. To assess the role of NK-NK homotypic interactions on the activation of NK cells, NK cells cultured in two different types of microwells, social microwells that allow contact-mediated multicellular interactions and lonesome microwells that inhibit cell-cell contact, were compared. Activation of NK cells by IL-2 was assessed by measuring phosphorylation of STAT-5, expression of CD25 and Ki-67. Immunofluorescence microscopy was performed to assess the levels of pSTAT-5, CD25, and Ki-67, and percentages of stained cells for each marker were examined and plotted. NK cells in social microwells expressed higher levels of all three markers than those in lonesome microwells, suggesting that homotypic contact-mediated interactions can enhance the activation of NK cells. Further analysis revealed that 2B4 is a key receptor mediating contact-mediated augmentation of NK cells in social miceowells by clustering and polarizing IL-2 receptor chains.
Interaction of TAPP adapter proteins with phosphatidylinositol (3,4)-bisphosphate regulates germinal centre response and development of autoimmunity (P1107)
Authors: Jayachandran Nipun , Landego Ivan , Marshall Aaron 
Published: January 2023

Abstract
Activation of PI3-kinase enzymes is essential for lymphocyte development and function. Active PI3Ks generate several types of D3 phosphoinositides, including PI(3,4,5)P3 and PI(3,4)P2. Tandem PH domain containing proteins (TAPPs) are adaptor molecules which bind specifically to PI(3,4)P2, but their functions in immune cells are largely unknown. We have performed studies on TAPP mutant mice, in which a mutation was introduced to the C terminal domain of both TAPP1 and TAPP2, to determine the effect of uncoupling TAPPs from PI(3,4)P2. We found that these TAPP KI mice exhibit elevated serum antibody levels and also develop autoantibodies and kidney pathology resembling that of lupus. TAPP KI mice generated elevated germinal center (GC) responses upon immunization with T-dependent antigen, suggesting an inhibitory role associated with TAPPs in lymphocytes. The GC B cell population was increased in frequency and showed reduced expression of apoptosis markers, and expansion of follicular helper T cell subset. We hypothesize that TAPP KI mice have disruption in a regulatory mechanism within the GC leading to sustained B cell activation and survival, and contributing to development of autoimmunity. TAPP KI B cells were found to be hyper-responsive to BCR cross-linking in vitro, and T cell responses are currently being assessed. Our goal is to define the B cell and T cell intrinsic signaling mechanisms enhanced as a result of disrupted inhibitory signaling mediated by TAPPs.
TNF-alpha receptors 1 and 2 contribute to the development of upper genital tract pathology following primary genital chlamydia muridarum infection in mice (P3200)
Authors: Murthy Ashlesh , Thomas Joshua , Manam Srikanth 
Published: January 2023

Abstract
Chlamydia is the leading cause of sexually transmitted disease worldwide and causes serious pathologies in the upper reproductive tract. In this study, we evaluated the contribution of TNF receptor 1 (TNFR1) and TNFR2 to upper genital tract pathogenesis following primary intravaginal chlamydial infection in mice deficient in TNFR1 (TNFR1 KO), TNFR2 (TNFR2 KO), or TNFR1 and 2 (TNFR1/2 DKO), and the corresponding C57BL/6J wild type mice. All groups (n=8) of mice displayed comparable chlamydial clearance following primary bacterial inoculation. The production of IFN-gamma, IL-5, TNF-alpha, and IL-1 beta in the genital tracts, draining iliac lymph nodes, and spleens on day 9 following inoculation were comparable in all the groups. Serum anti-Chlamydia total Ab, IgG2c, and IgG1 response on days 14 and 60 following primary inoculation were comparable in all the groups. On day 80 after primary incoluation, TNFR1 KO, TNFR2 KO, and TNFR1/2 DKO mice displayed significantly reduced hydrosalpinx (6 %, 44%, and 0% oviducts, respectively), when compared to C57BL/6J mice (75% oviducts). Anti-Chlamydia antibody response and bacterial clearance following secondary intravaginal C. muridarum infection was comparable in all groups of mice. Collectively, these results suggest that TNFR1 and TNFR2 do not contribute significantly to chlamydial clearance, but both TNFR1 and TNFR2, with a dominant role for TNFR1, contribute to the UGT pathology following primary genital chlamydial infection.
The exposure of the lungs to both fungi and non-replicating virus-like particles, elicits immune imprinting to provide exquisite protection against subsequent influenza virus challenge (P3214)
Authors: Richert Laura , Rynda-Apple Agnieszka , Wiley James , Harmsen Ann , Douglas Trevor , Harmsen Allen 
Published: January 2023

Abstract
The pulmonary delivery of both virus-like particles (VLPs) (which bear no antigenic similarities to respiratory pathogens), and the opportunistic fungi, Pneumocystis murina, acted to prime the lungs of mice, thus facilitating heightened and accelerated primary immunity to high-dose influenza challenge. These responses were characterized by accelerated antigen processing by alveolar macrophages (AM’s) and dendritic cells (DC’s) and DC trafficking to the local tracheobronchial lymph node (TBLN). Additionally, CD11c+ cells which had been directly exposed to VLPs were necessary in facilitating the observed enhanced viral clearance. The repopulation of CD11c+ DC’s and AM’s from Ly-6Chi myeloid precursors relied on the expression of CCR2, and in the absence of efficient Ly-6Chi cell trafficking in CCR2 knockout mice, or via antibody depletion, the protection afforded by both Pneumocystis- and VLP-exposure was lost. Thus, immune imprinting in the lung by Pneumocystis infection, or VLP-exposure allowed for accelerated influenza-specific primary immune responses in both the lung and TBLN via Ly-6C- or CCR2-dependent mechanisms, respectively. Importantly, both models resulted in enhanced viral clearance and reduced collateral damage.
Human chorionic gonadotropin and luteinizing hormone, but not progesterone, contribute to the initiation of a tolerogenic immune response in pregnancy (P3216)
Authors: Schumacher Anne , Heinze Kristina , Spörke Dominique , Poloski Eileen , Zenclussen Ana 
Published: January 2023

Abstract
The survival of the fetus in the maternal uterus is ensured by both intensive hormonal changes and a protective maternal immune response towards the fetus. We have recently reported that human Chorionic Gonadotropin (hCG) facilitates the migration of Treg to the fetal-maternal interface and supports their tolerogenic function. The role of hormones in the establishment of an antigen-specific immune response is however not well understood. Here, we investigated the effect of pregnancy hormones on the maturation state of DCs and their functionality in a model of spontaneous abortion due to disturbed tolerance. Pregnant females were treated either with hCG, Luteinizing Hormone (LH), Progesterone (P) or PBS. Pregnancy outcome and the number of total and mature DCs was evaluated in the periphery and in the uterus. The effect of hCG on DC maturation and functionality was assessed in vitro. hCG and LH application totally prevented fetal rejection while P did not. The protective effect of hCG and LH was associated with a reduction in the number of total and mature DCs in the periphery and at the fetal maternal interface. By contrast, P did not affect DC phenotype. In vitro, hCG impaired the maturation process of DCs confirming our in vivo data. Our results highlights the importance of hormones in regulating the initiation of the adaptive immune response that allows the transient acceptance of paternal antigens and thus importantly contributes to pregnancy success.
Acute non-inflammatory epithelial death plays a role in intrinsic defense against enteric bacteria (P3064)
Authors: Chang Sun-Young , Lee Se-Na , Ko Hyun-Jeong , Kweon Mi-Na 
Published: January 2023

Abstract
Host cell death is critical to the fate of bacterial infection, to host innate immune responses, and to disease outcome. In this study we found that Shigella organisms could invade the terminal ileum of the small intestine in mice by 1 hour of early phase infection and be rapidly cleared within 24 hours even though Shigellae are considered human-specific pathogens. Early phase events after oral infection showed epithelial shedding, degranulation of Paneth cells, and cell death in the intestine without any signs of inflammation. Dead products including DNA and intracellular organelles trapped Shigellae and damaged tissue rapidly recovered within a day. These acute responses occurred with both human-specific pathogens such as Escherichia coli and with such murine pathogens as Salmonella typhimurium. Of interest, human intestine also shared this intrinsic defense. Here we propose a general intrinsic mechanism that repels the initial introduction of entero-pathogenic bacteria by sacrificing host cells.
Mast cells promote the migration and phenotypic conversion of myeloid derived suppressor cells in parasitic infection. (P3080)
Authors: Martin Rebecca , Saleem Sheinei , Conrad Daniel 
Published: January 2023

Abstract
Myeloid derived suppressor cells (MDSCs) are mainly known for their immunosuppressive properties in the tumor microenvironment. However, we have shown that in the context of parasitic infection, MDSCs play a beneficial role. In fact, mice infected with Nippostrogylus brasiliensis (Nb) and adoptively transferred (AT) with MDSCs have enhanced parasite clearance. This immunosupportive activity of MDSCs is limited to the granulocytic CD11b+Gr-1+Ly6G+ subset of MDSCs. Upon AT, MDSCs preferentially migrate to the liver, where they interact with resident MCs in the portal vein. The liver is the proposed site of proliferation, activation of MDSCs, and skewing towards the beneficial granulocytic population. Furthermore, MC deficient mice are unable to clear Nb albeit the presence of MDSCs and have reduced trafficking of MDSCs to the liver. In separate neoplastic studies, we have demonstrated that MC histamine release is responsible for the activity of MDSCs. To confirm this in our parasitic model, we utilized cetirizine, a histamine receptor 1 (HR1) antagonist. In fact, HR1 blockade abrogated MDSC activity and decreased parasite clearance. Furthermore, we observed decreased MDSC migration to the liver, proliferation, and conversion towards Ly6G+ population. Taken together, our studies indicate that in the absence of MCs as well as inhibition of histamine signaling, MDSCs are unable to exert their immunostimulatory properties and enhance parasite clearance.
The roles of CD36 in host defense and homeostasis (P3052)
Authors: Stefanov Emily , Kearney John 
Published: January 2023

Abstract
The immune recognition of polysaccharide antigens is vital in both host defense against pathogens and general housekeeping functions in the body. CD36 is a Type-BI scavenger receptor expressed by multiple cell types including APCs and subsets of B cells. CD36 recognizes various endogenous and pathogen-derived ligands, including β-glucans, lipoteichoic acid, and oxidized phospholipids. Therefore, we hypothesized that CD36 would have important roles in the recognition of and host defense against pathogens bearing these ligands. We observed that mice lacking CD36 show increased susceptibility to infection by S. pyogenes and A. fumigatus, which express these ligands and had a decreased ability to generate anti-N-acetyl-D-glucosamine (GlcNAc) antibodies in response to immunization with S. pyogenes. CD36-/- APCs also exhibit a reduced capacity to phagocytose these microorganisms in vitro and in vivo. Additionally, we observed that mice lacking CD36 have an exacerbation of A. fumagatus-induced allergic airway disease and an accumulation of proinflammatory cells in the alveolar spaces of the lung. We propose that this is due to the reduced ability to clear A. fumigatus conidia and the diminished capacity of CD36-/- phagocytes to clear apoptotic cells. Our data suggest that CD36 on both B cells and APCs plays important roles in the development of anti-GlcNAc antibody responses and the clearance of allergens to modulate the development of allergic airway disease in mice.
Combined targeting of exposed phosphatidylserine, CD47 and CD54 on human pancreatic tumor cells in a mouse xenograft model of human pancreatic cancer (P4455)
Authors: Ma Shihong , Thorpe Philip , Vitetta Ellen , Meyer Jeffrey 
Published: January 2023

Abstract
Phosphatidylserine (PS) is normally intracellular but becomes exposed on the luminal surface of vascular endothelium in tumors and can also be expressed on tumor cells themselves. Studies have shown that targeting PS with an antibody enhances the antitumor effect of chemotherapy or radiation on several tumors, and induces antitumor immunity in mice. Combination of this anti-vascular and antitumor antibody with an antibody (anti-CD47) that blocks the “don’t eat me” signal CD47 commonly expressed on human tumors may lead to synergistic antitumor activity. Similarly, synergistic antitumor activity might be obtained by combining the PS-targeting antibody with anti-CD54, an antibody that has been demonstrated to have pan antitumor activity in xenograft models and is safe in humans. Our objective is to evaluate combinations of these antibodies in a xenograft model of human cancer. Studying these combinations requires determining the expression of CD47 and CD54 on several cancer cell lines. To this end, MDA-MB-231 (breast), NCI-H157 (lung), Du145 (prostate), PANC-1 and BxPC-3 (pancreatic) were analyzed for surface expression of CD47 and CD54. All five cell lines expressed CD47 (&gt;87% of cells positive). All tested cell lines were also highly CD54 positive (&gt;84% of cells positive) except for MDA-MB-231 (~50% positive). The effects of anti-PS antibody in conjunction with anti-CD47 or anti-CD54 on tumor growth are currently being analyzed in SCID mice bearing BxPC-3 pancreatic tumors.