bluuebunny/crossref_metadata_2025 huggingface dataset:
Innate cytokines associate with exosomes in plasma of HIV-1+ individuals (P6205)
Authors: Johnson Kateena , Chu Jane , Huang Ming , Powell Michael , Villinger Francois , Bond Vincent
Published: January 2023

Purpose: Our group and others have demonstrated a potential role for exosomes in HIV pathogenesis. Exosomes secreted from HIV-infected cells were shown to contain coreceptors for HIV. We have shown HIV Nef-containing exosomes from HIV-infected cells induced apoptosis in bystander CD4+ T cells. This suggests that exosomes from HIV-infected cells could drive CD4 depletion and immune activation contributing to HIV pathogenesis. Thus, exosomes from HIV-1+ viremic individuals were investigated for protein content. Methods: Exosomes were isolated from plasma of ARV naïve, HIV+ and negative control individuals by differential centrifugation followed by iodixanol density gradient ultracentrifugation. Biochemical characterization included AChE activity, flotation in iodixanol gradients, and Western analysis for exosomal markers. Plasma and purified exosomes were also tested for levels of 30 cytokines and chemokines by multiplex assay. Results: The concentrations of 10 innate cytokines (IL-15, IP-10, FasL, CD40L, IFNα, IFNβ, MIP-1α, MIP-1β, TNFα, and ICAM-1) were found to be associated with and significantly elevated in exosomes from HIV+ individuals relative to negative controls, suggesting a contributing role in generalized chronic immune inflammation. Conclusion: This is the first demonstration of innate cytokines in exosomes of HIV+ individuals. It suggests a potential mechanism for HIV-1 driven immune modulation and propagation of HIV via activation of bystander target cells.
Cell-intrinsic HIV-1 immune responses in conventional dendritic cells from HIV-1 elite controllers (P6171)
Authors: Martin-Gayo Enrique , Hickman Taylor , Pimenova Dina , Pereyra Florencia , Rosenberg Eric , Lichterfeld Mathias , Yu Xu
Published: January 2023

Introduction: Recent data suggest that in most HIV-1-infected individuals, cell-intrinsic immune responses of conventional dendritic cells (cDC) are blocked by the host proteins Samhd1 and Trex1. Elite controllers (EC) control HIV-1 replication in the absence of treatment, but immune defense mechanisms in these patients are not well understood. Here, we investigated cell-intrinsic immune responses to HIV-1 in these specific patients. Methods: PBMC from EC, untreated chronic progressors (CP), HAART-treated and HIV-1 negative subjects were ex vivo infected with HIV-1. Expression of viral replication products, type I interferons, Samhd1 and Trex1 were analyzed by qPCR. Results: cDC from HIV-1 negative persons were moderately susceptible to HIV-1, while cDC from EC and CP supported HIV-1 replication very weakly. However, in CP, HIV-1 replication was blocked at the level of early reverse transcription, likely as a result of high-level Samdh1 expression, while In EC, reverse transcription was unaltered, and restriction of viral replication mostly occurred at the level of integration. Functionally, these altered patterns of viral restriction in cDC from EC were associated with increased cellular activation, secretion of type I interferons and improved abilities to prime T cell responses. Conclusion: cDC from EC can mount cell-intrinsic immune responses against HIV-1, which may support the generation of highly effective HIV-1-specific T cell responses in these patients.
Lysine dimethylation in the BH4 domain of Bcl-2 and functional significance (P1306)
Authors: Li Wenqing , Xiao Zhen , Ravichandran Sarangan , Collins Jack , Veenstra Timothy , Durum Scott
Published: January 2023

Bcl-2 is an anti-apoptotic protein that can protect developing and mature T cells against a variety of apoptotic signals. The Bcl-2 family of proteins, compromised of anti-apoptotic as well as pro-apoptotic members, plays key roles in apoptosis; the balance of these members determines cell survival. Anti-apoptotic Bcl-2 contains four conserved Bcl-2 homology (BH) domains and the BH4 domain is indispensable for its ability to suppress cell death. Using mass spectroscopy to analyze a murine T cell line, we identified dimethylation of two lysine residues in the BH4 domain: K17 and K22. Mutation of K22 (but not K17) to alanine abrogated Bcl-2 binding to pro-apoptotic proteins Bax, Bak, Bad, Bim and Puma and greatly reduced Bcl-2 anti-apoptotic function in cytokine withdrawal. This assigns functional importance to K22 (which is conserved across species and other anti-apoptotic members) and suggests that dimethylation could affect its function. K22, based on structural models, appears to be involved in stabilizing peptide secondary structure, rather than directly interacting with pro-apoptotic proteins. We developed a monoclonal antibody that recognizes dimethylated K22 in the Bcl-2 sequence and detected expression in murine B cells (but not thymocytes). This suggests that Bcl-2 dimethylation may regulate B cell survival.
CD56 is a legitimate immune receptor regulating T cell and NK cell effector function, and its expression level predicts successful aging (P1324)
Authors: de Vallejo Abbe , Griffin Patricia , Montag David , Nussbaum Ryan , Gunawardema Naomi , Studenski Stephanie
Published: January 2023

CD56 is a classical marker of NK cells, but its direct relevance to the elaboration of NK cell effector function is not known. In two cohort studies of aging, we have found that high levels of CD56 expression on both NK cells and T cells are highly correlated with unimpaired cognitive and physical ability among older adults aged ≥65 years. In multivariate analysis of collated data on clinical and laboratory assessments of health and performance, we identified CD56 as a key immunologic predictor of successful aging, an emerging physiologic and clinical construct pertaining to the maintenance of functional independence in old age despite a history of disease and concurrent comorbid conditions. In biological assays using primary T and NK cells, and various immortalized T/NK cell lines, ligation of CD56 alone was sufficient to induce activation of NK cells, and of T cells in a TCR-independent manner. CD56 triggering effectively elicited expression of activation antigens such as CD69 and CD25, and the production of various cytokines including IL-2, IL-4, and IFN-γ. CD56-mediated activation of both T cells and NK cells involved LAT phosphorylation. Clearly, CD56 is a functional immune receptor capable of effecting proximal and distal cellular outcomes. TCR-independent CD56-driven activation of T cells in particular is consistent with the concept of beneficial immune remodeling that has been proposed to underlie healthy aging and long-term survivorship.
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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 (>87% of cells positive). All tested cell lines were also highly CD54 positive (>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.
CD4+ helper T cells and immunity to therapeutic proteins in gene therapy (P4431)
Authors: Bremer William , Campbell Katherine , Rodino-Klapac Louise , Goodchild Laurie , Griffin Danielle , Montgomery Chrystal , Walker Christopher
Published: January 2023

Adeno-associated (AAV) vectors are being assessed for gene replacement therapy in humans. Proteins encoded by the vector transgene are potentially foreign. Strategies to temper destructive immune responses may be required to achieve a therapeutic effect, particularly in diseases like Duchenne Muscular Dystrophy where the defective gene to be replaced has a large frame-shifting deletion. Here we studied the impact of immunity on expression of enhanced green fluorescent protein (eGFP) in AAV-transduced skeletal muscle of rhesus macaques. Antibodies to eGFP developed rapidly after AAV vector delivery to the tibialis anterior muscle. Cellular immunity was variable. In some animals, strong CD4+ helper and CD8+ cytotoxic T cells were detected in blood 2 weeks after vector administration. In others, low frequency responses were not detected until week 5-6. Regardless of the pace of the response, eGFP was cleared or substantially diminished in muscle after week 6. Antibody-mediated depletion of CD4+ T cells before AAV vector treatment delayed antibody and CD8+ T cell immunity and facilitated robust expression of eGFP persisted through day 42. Some positive muscle fibers were still visible at month 10, an unexpected finding that suggested an inability to sustain destructive cellular immune responses initially primed without CD4+ T cell help. The capacity for recall of eGFP-CD8+ T cell responses upon administration of a serologically distinct AAV vector is now being assessed.
Reciprocal interaction between NK cells and DCs regulates the Th17 response by controlling the innate IFN-γ/IL-27 axis (P4067)
Authors: Caspi Rachel , Chen Jun , Silver Phyllis , Horai R. , Mattapallil Mary , Zhou Ru , Chong WaiPo
Published: January 2023

Experimental autoimmune uveitis (EAU) is a model for human uveitis. IFN-γ deficient mice develop enhanced EAU with elevated Th17 responses. This protective effect of IFN-γ appears at odds with the ability of IFN-γ-producing Th1 cells to induce EAU. We previously showed that it is innate IFN-γ that is protective, whereas adaptive IFN-γ is pathogenic. We now demonstrate that NK cells, a major cellular source of innate IFN-γ, interact with DCs in draining lymph nodes of EAU-challenged mice to ameliorate disease by controlling the innate IFN-γ/IL-27 axis. After immunization, both NK cells and DCs were recruited to the draining lymph nodes, with recruitment of NK cells being at least partially dependent on DCs and CXCR3. Infusion of IFN-γ-sufficient WT NK cells into IFN-γ-/- recipients reduced their EAU scores and enhanced production of IL 27, a suppressor of the Th17 response, from DCs of these mice. In vivo neutralization of IL-27 abolished the protective effect of WT NK cells on EAU. In vitro experiments revealed that mature DCs induced NK cells to produce IFN-γ, which in turn caused DCs to produce IL-27. IL-27 then stimulated NK cells to produce more IFN-γ, suggesting the presence of a positive feedback loop. IL-27 also promoted generation of IL-10-producing Tr1-like cells. Our results suggest that the magnitude of the Th17 response, and consequently the severity of disease, is controlled by the interaction between NK cells and DCs through the IFN-γ/IL-27 axis.
FAS-associated death domain and tripartite motif-containing protein 21 negatively regulate virus-induced interferon production (P6126)
Authors: Nandu Suruchi , Chandra Neha , Winoto Astar
Published: January 2023

While induction of type I Interferon (IFN) during the host antiviral response is imperative for efficient viral clearance, regulation of this response is crucial for immune system homeostasis. We have reported that FADD, a well characterized apoptotic protein, is involved in negatively regulating RNA virus-induced IFN-α production through a novel interaction with the E3 ubiquitin ligase, Tripartite Motif-Containing Protein 21 (TRIM21). Interaction between FADD and TRIM21 cooperatively represses IFN-α activation in Sendai virus infected cells. This occurs through TRIM21 targeted ubiquitin-mediated degradation of transcription factor IRF7. Recently, it was also determined that FADD and TRIM21 can be found in 2 different complexes within cells: one containing FADD/TRIM21/IRF7 and a second containing FADD/TRIM21/RIP1 (receptor-interacting protein 1). We hypothesized that TRIM21 may have different functions dependent on complex composition and that its activity could be regulated via its autoubiquitination status. Interaction of TRIM21 with FADD yields increased autoubiquition while interaction with RIP1 yields decreased autoubiqtuitination suggesting a regulation mechanism. However, studies involving mutant forms of TRIM21 which lack autoubiquitination demonstrate that autoubiquitination status does not regulate TRIM21’s ability to target IRF7 for degradation. We are currently investigating the function of TRIM21 in other FADD/RIP1 dependent processes like cell death.
Bovine natural killer cells are infected by BVDV and the infection results in changes in phenotype and activation markers (P6128)
Authors: Darweesh Mahmoud Darweesh , Rajput Mrigendra , Lebedev Max , Young Alan , Braun Lyle , Chase Christopher
Published: January 2023

Bovine viral diarrhea virus infection results in immunosuppression and persistence infection of fetuses. Understanding the interaction between BVDV and NK cells is important to understand the ability of the virus to establish its infection especially through placental barrier to generate persistently infected animals. In this study, we demonstrated that BVDV is able to infect bovine NK cells by detecting the presence of BVDV RNA level up to 24 hours post infection without any increase in RNA levels over time. We also confirmed that although BVDV viral RNA was present, there was neither infectious virus release at any time post infection nor decrease in cell viability due to infection. Using immunofluorescence staining of NK cells with a mAb specific for BVDV E2 protein, we demonstrated the presence of BVDV E2 proteins in NK cells. The effect of BVDV strains that differ in its virulence or biotype on NK activation marker CD25 and phenotype markers CD2, NKp46 and the granule protein granulysin was determined. Highly virulent BVDV strains significantly decreased surface expression of each of these markers with a slight increase in the total number of cells expressing the markers 24 hour post infection. On the other hand less virulent strains increased significantly the percent of expression but with a slight decrease in the number of cells expressing the markers.
Prenatally acquired vitamin A deficiency alters innate immune responses to human rotavirus in a gnotobiotic pig model (P6145)
Authors: Saif Linda , Chattha Kuldeep , Kandasamy Sukumar , Siegismund Christine , Vlasova Anastasia
Published: January 2023

We examined how prenatally acquired vitamin A deficiency (VAD) modulates innate immune responses and human rotavirus (HRV) vaccine efficacy in gnotobiotic (Gn) pigs. The VAD and vitamin A sufficient (VAS) Gn pigs were vaccinated with attenuated HRV (AttHRV) with or without concurrent oral vitamin A supplementation (100,000IU) and challenged with virulent HRV (VirHRV). Regardless of vaccination status, the numbers of conventional and plasmacytoid dendritic cells (cDCs and pDCs) were higher in VAD vs. VAS piglets pre-challenge, but decreased substantially post-challenge. We observed significantly higher frequency of CD103 (integrin αEβ7) expressing DCs in VAS vs. VAD piglets post-challenge, indicating that VAD may interfere with homing (including intestinal) phenotype acquisition. Post VirHRV challenge, we observed longer/more pronounced diarrhea and higher VirHRV fecal titers in non-vaccinated VAD piglets. Consistent with higher VirHRV shedding titers, higher IFNα levels were induced in control VAD vs. VAS piglet sera at post-challenge day (PCD)2. HRV-stimulated mononuclear cells (MNCs) isolated from spleen and blood of VAD pigs pre-challenge also produced more IFNα. In contrast at PCD10, we observed reduced IFNα levels in VAD pigs that coincided with decreased TLR3+ MNC frequencies. Thus, prenatal VAD caused an imbalance in innate immune responses and exacerbated VirHRV infection, whereas vitamin A supplementation failed to compensate for these VAD effects.
Corneal-derived dendritic cells are critical in T cell expansion after HSV-1 ocular infection (P6102)
Authors: Buela Kristine-Ann , Hendricks Robert
Published: January 2023

Corneas possess resident DC (cornea-resident DC), and infiltrating DC migrate to the draining lymph nodes (DLN) following HSV-1 corneal infection (cornea-derived DC). The relative contribution of these two DC populations and DLN-resident DC to CD4+ and CD8+ T cell expansion in DLN following HSV-1 corneal infection is unknown. Local depletion of cornea-resident DC in CD11c-DTR mice did not affect T cell expansion in the DLN. Depletion of cornea-derived DC through 3 days post infection (dpi) abrogated early (3 dpi) CD8 T cell expansion (BrdU incorporation), but only reduced CD4 T cell expansion by 50%. Systemic DC depletion through 3 dpi completely abrogated expansion of both CD4 and CD8 T cells at 3 dpi. Depletion of cornea-derived DC through 7 dpi abrogated expansion of both CD4 and CD8 T cell at 7 dpi. Cornea-derived DC showed greater expression of CD70, CD80, and CD86 relative to DLN-resident DC. Blocking CD70 co-stimulation significantly reduced CD8 T cell survival and numbers, but did not affect their proliferation. We conclude that: (i) DC are required for expansion of both HSV-specific CD4+ and CD8+ T cells; (ii) up to 3 dpi CD8 T cells absolutely require HSV-1 presentation by cornea-derived DC possibly due to their enhanced costimulation, whereas DLN-resident DC can stimulate CD4 T cells (presumably with free virus that reaches the DLN from the cornea); (iii) after 3 dpi expansion of both CD4+ and CD8+ T cells requires transport of viral antigens by cornea-derived DC.
IL-33 induces nuocytes and modulates liver injury in viral hepatitis (P6112)
Authors: Liang Yuejin , Jie Zuliang , Hou Lifei , Aguilar-Valenzuela Renan , Vu David , Soong Lynn , Sun Jiaren
Published: January 2023

Our aim was to investigate the role of IL-33, the damage-associated molecular pattern (DAMP) molecule, in acute viral hepatitis. C57BL/6 mice were i.v. infected with a recombinant adenovirus. IL-33 and its receptor ST2 expression increased steadily in the liver in the first week of the infection. Treatment of exogenous IL-33 resulted in a great decrease in the serum alanine aminotransferase (ALT) levels and the number of Councilman bodies. Attenuated liver injury by IL-33 correlated with an increase in T regulatory (Treg) cells but with a decrease in macrophages, dendritic cells and NK cells in the liver. IL-33 enhanced both type 1 (IL-2, IFN-γ) and type 2 (IL-5, IL-13) immune responses in infected mice. However, IL-33 inhibited TNF-α in hepatic T cells and macrophages, and significantly reduced TNF-α levels in the liver. In addition to direct effects of IL-33, we found that IL-33 strongly induced nuocytes in the liver and spleen of infected mice. When co-cultured with nuocytes, hepatic T cells and macrophages expressed lower levels of TNF-α. In addition, the adoptive transfer of nuocytes led to lower serum ALT levels in the Ad-infected recipient mice. In conclusion, this study reveals that IL-33 acts as a potent immune stimulator and a hepatoprotective cytokine in acute viral hepatitis. Its direct immunoregulatory functions and ability to induce nuocytes further suggest to us that it may be a potentially promising therapeutic candidate for the management of viral hepatitis.
Common activation mechanism of beta 2 integrins (P5073)
Authors: Yuki Koichi , Sen Mehmet , Springer Timothy
Published: January 2023

Beta 2 integrins are α/β heterodimeric adhesion molecules on leukocytes consisting of αLβ2, αMβ2, αXβ2 and αDβ2. They undergo significant conformational changes to bind to their ligands at the domain called the α I domain of the α subunit. It has been postulated that the domain called the β I domain of the β subunit undergoes conformational changes and crosstalks with the α I domain in favor for ligand binding. However, the regions important for the β I domain activation have not been determined yet. Our unpublished and published αXβ2 structures suggested that the α1 helix of the β I domain was important in the activation of the β I domain. To examine the importance of the α1 helix, alanine scanning mutagenesis was performed on this region and transient transfectants in 293T cells were used for the experiments. Our β2 V124A and L132A mutations were constitutively active in all αL, αM, and αX integrins as demonstrated by ligands’ binding (αLβ2 to ICAM-1, αMβ2 to fibrinogen, ICAM-1, and iC3b, αXβ2 to iC3b) and activation-sensitive epitope exposures (KIM127, m24 and MEM148) even without activation. On the other hand, β2 L127A and L135A mutations were deactivating in all αL, αM and αX integrins as shown by diminished ligands’ binding and activation-sensitive epitope exposures even in an activating condition. Our data suggested that b2 integrins shared the common activation pathway of the β I domain, which would subsequently relay to the αI domain.
Permission of pathogen expansion inside a specialized DC by a co-inhibitory pathway dictates T cell response (P4249)
Authors: Fu Yang-Xin , Yang Xuanming
Published: January 2023

B and T lymphocyte attenuator (BTLA), a co-inhibitory receptor, is required to suppress T cell mediated immunity. We demonstrated that cross-linking BTLA by herpesvirus entry mediator (HVEM) suppresses T cell responses against Listeria, while blocking BTLA enhances the T cell response at not only primary but also memory phase. Unexpectedly, BTLA-/- mice have significantly lower CD8+ T cell effector and memory cells while paradoxically being more resistant to Listeria monocytogenes infection. Although there is no reduction in listerial entry into BTLA-/- CD8+ DCs, intriguingly, Listeria fails to expand within these DCs. We show that BTLA signaling is essential for limiting Fas/FasL-mediated suppression of Listeria expansion within host CD8+ DCs, which more effectively alert adaptive immune cells. This study provides new evidence that a co-inhibitory pathway for T cells can increase overall T cell responses by permitting pathogen proliferation within specialized DCs.
A role for IL-10 in efferocytosis of apoptotic neutrophils and resolution of lung inflammation produced by STAT1-regulated lung MDSC-like cells (P4255)
Authors: Arora Meenakshi , Poe Stephanie , Ray Anuradha , Ray Prabir
Published: January 2023

Bacterial pneumonia remains a significant burden worldwide. Little is known about cellular and molecular mechanisms in the lung tissue that remove apoptotic neutrophils to prevent collateral tissue damage during bacterial infection. In a mouse model of bacterial infection with Klebsiella pneumoniae, we observed a role for IL-10, produced in a highly orchestrated fashion by tissue-resident cells, in resolution of pulmonary inflammation and recovery of mice post-infection. While IL-10-/- mice cleared bacteria, they displayed increased morbidity with progressive weight loss and persistent lung inflammation. The major source of tissue IL-10 was CD11b+Gr1intF4/80+ cells resembling myeloid-derived suppressor cells (MDSCs) that accumulated with a delayed kinetics in the lung tissue after infection and efficiently efferocytosed apoptotic neutrophils. However, increased bacterial burden handicapped this function by decreasing the ratio of the efferocytic MDSC-like cells:inflammatory neutrophiils in the lung. To overcome this undesirable outcome, we took advantage of functional antagonism between STAT1 and STAT3. In STAT1-/- mice, the excessive neutrophil infiltration in the lungs elicited by a high dose of K. pneumoniae was attenuated with concomitant increase in the frequency of the MDSC-like cells that expressed increased pSTAT3 levels. Thus, inhibiting STAT1 may be a novel therapeutic strategy to address inefficient resolution of bacterial pneumonia.
Critical role of TRIF in modulating innate and adaptive responses during Citrobacter rodentium infection. (P4037)
Authors: Gurung Prajwal , Kanneganti Thirumala
Published: January 2023

Citrobacter sp. is a gram negative coliform facultative bacteria that is mostly associated with the inflammation of the gut and is widely studied as the mouse model organism for studying colitis. We previously showed that Citrobacter rodentium induces non-canonical inflammasome activation through a TRIF-dependent mechanism, however the exact role of TRIF during C. rodentium induced colitis is still unclear. Herein, we have used C. rodentium induced colitis model to study the role of TRIF in vivo and showed that TRIF plays a critical role in protection during C. rodentium induced colitis. Absence of TRIF results in increased bacterial burden and morbidity, which is dramatically increased when TRIF is absent in a Myd88-deficient background. TRIF-deficiency resulted in increased neutrophil numbers in the spleen. Similarly, dendritic cells frequency and numbers were dramatically increased and displayed activated phenotype. T cell frequencies and numbers were similar, however TNF-alpha producing CD4+ and CD8+ T cells were dramatically reduced. Further studies are being carried out in the lab to understand how TRIF regulates these various populations and functions of the innate and adaptive cells.