bluuebunny/crossref_metadata_2025 huggingface dataset:
Innate defence regulator peptide 1018 protects mice in the Pseudomonas aeruginosa acute lung infection model (P4045)
Authors: Hilchie Ashley , Magrangeas-Janot Laure , Wuerth Kelli , Hancock Robert
Published: January 2023

Host defence peptides (HDPs) are small peptides with antibacterial and immunomodulatory properties that represent a novel approach to anti-infective therapy. Innate defence regulator (IDR) peptides, such as IDR-1018, are synthetic variants of HDPs that are more potent and less toxic than their naturally-occurring counterparts. IDR-1018 kills bacteria, exhibits potent chemokine activities, and decreases LPS-induced TNF-α release. The goal of the current study was to determine whether IDR-1018 protects mice in the Pseudomonas aeruginosa acute lung infection model. To this end, CD1 mice were given IDR-1018 (4 mg/kg) or the saline control intravenously 4 h prior to infection. Mice were infected intranasally with P. aeruginosa (1 x 10^6). Mice were sacrificed 18 h post infection, and blood and bronchoalveolar lavage fluid (BALF) was collected. P. aeruginosa CFUs and leukocyte counts in the BALF were determined. Cytokine concentrations (TNF-α, MCP-1, KC, and IL-6) in the blood and BALF were quantified by ELISA. Our data showed that IDR-1018-treated mice had fewer CFUs and neutrophils and more monocytes in the BALF, and lower concentrations of proinflammatory cytokines in the BALF and in the blood than did saline-treated mice. These results suggest that IDR-1018 may have potential for the treatment of acute lung infections. Future studies aim to optimize the delivery strategy by identifying a delivery vehicle that enhances the half life and ease of peptide delivery.
Celecoxib induces apoptosis, ER stress, and cyclooxgenase-2 expression on lung cancer cells (P2090)
Authors: Kim Ja-Young , Kim Hae jin , Choi Yunock , Park Ga Bin , Kim Seonghan , Hur Dae Young , Kim Yeong Seok
Published: January 2023

Cyclooxygenase-2 (COX-2) is an enzyme that is inducible by various stimuli such as inflammation. COX-2 expression is increased in a variety of human premalignant and malignant tumors. Celecoxib is a selective inhibitor of COX-2 that have been shown to inhibit cancer cell growth and exert anti-proliferative and pro-apoptotic effects on various cancer cell lines. In this study, we investigated that celecoxib induced apoptosis of lung cancer cell lines, A549 and H460. Increase of apoptotic cells was investigated on celecoxib treated lung cancer cell lines. Cell proliferation was decreased and sub G0 peak was increased. We also observed that celecoxib induced activation of sensor proteins involved in endoplasmic reticulum (ER) stress. Expression of CHOP was induced after treatment of celecoxib on lung cancer cells on time-dependent and dose-dependent manners using RT-PCR and western blot. However, salubrinal, specific inhibitor of eIF-2 alpha phosphatase, did not block CHOP expression and cell death after celecoxib treatment. Paradoxically, celecoxib stimulated COX-2 mRNA and protein on A549, but indomethacin, inhibitor of COX-1 and COX-2 did not induce COX-2 expression. Taken together, we suggest that celecoxib induces apoptosis and ER stress on lung cancer cell line.
α-Galactosylceramide-loaded tumor cell vaccine promotes NKT-dependent protective immunity against multiple myeloma (P2096)
Authors: Choi Inhak , Hong Sungyoul , Lee Hyeonsoo , Jeon You-Kyoung , Lee Eungchang
Published: January 2023

Despite progress in the treatment of multiple myeloma (MM), this disease still remains fatal in the majority of patients. Thus, new immunotherapeutic approaches are required to eradicate minimal residual myeloma cells. Tumor cells can be a source of tumor-specific antigens for immunotherapy. However, tumor cells have been weak to promote tumor-specific immune responses and maintain the tumor-specific responses with large amounts of tumor burden in vivo. In this study, we evaluated and compared the efficacy of the vaccination of autologous myeloma cells loaded with artificial ligand for T cell receptor of NKT cells, α-galactosylceramide (αGC), in the myeloma mouse model. Our results showed that αGC-loaded MOPC315BM myeloma cell vaccines efficiently retarded tumor growth, induced tumor regression against established tumor, and protected surviving mice from tumor rechallenge. The therapeutic responses were associated with an induction of strong humoral immune responses including anti-myeloma cell antibodies and cellular immune responses including myeloma-specific CD8+ cytotoxic T lymphocytes and memory T cells. In addition, we found that regulatory T cells were significantly decreased in mice receiving αGC-loaded myeloma cell vaccines. Thus, our results demonstrate that αGC-loaded myeloma vaccine efficiently promote NKT-dependent anti-tumor immunity in our study.
Utilizing dual-specific CD4+ T cells in combination with oncolytic virotherapy for treatment of cancer (P2118)
Authors: VanSeggelen Heather , Hammill Joanne , Bassett Jennifer , Rabinovich Brian , Bramson Jonathan
Published: January 2023

Engineering T cell specificity to direct them against tumor cells utilizing chimeric antigen receptors (CARs) has emerged as an exciting technique for adoptive T cell therapy. The effectiveness of adoptive transfer therapies can be limited by a number of factors, including ability of T cells to engraft, the degree to which they infiltrate the tumor, and the immunosuppressive nature of the tumor. Oncolytic viruses (OVs) are capable of overcoming these limitations, and can serve as effective booster vaccines. In an effort to capitalize on these features, we have generated dual-specific T cells that bear TCRs specific for the OV, and CARs specific for the tumor. Using murine tumor models, we have observed significant boosting capacity of adoptively transferred dual-specific CD4+ or CD8+ T cells to tumor-bearing hosts in response to infusion with OV. Boosting with OV also dramatically influenced the distribution of the CAR-T cells, driving the cells to proliferate throughout the body. We now seek to examine the utility of dual-specific CD4+ T cells in adoptive transfer therapies. We have found that CAR-engineered CD4+ T cells are capable of mediating anti-tumor immunity, and are investigating the combination of these cells with OV to further enhance their anti-tumor efficacy. Given the promising clinical outcomes of both CAR T cells and OVs, we believe our combinatorial approach will provide a clinically feasable strategy to maximize the therapeutic potential of both therapies.
ICOS signaling in CD8+ CTL contributes to the antitumor efficacy (P2122)
Authors: Fu Tihui , Peng Weiyi , He Qiuming , Hwu Patrick , Sharma Padmanee
Published: January 2023

The expression of Inducible costimulator (ICOS) on CD8+ CTL and its role in antitumor immunity remains largely unknown. To address this issue, we crossed ICOS-deficient mice with pmel-1 transgenic mice to generate pmel1/ICOS-/- mice. The loss of ICOS in pmel-1CD8+ CTL cells did not affect the expression of other T cell activation markers (e.g. CD25 and CD69) but led to a significant decrease in IFN-gamma production and killing of target tumor cells. Similarly, adoptive transfer of pmel-1 cells into ICOS-ligand knockout mice showed impaired cell proliferation and decreased expression of IFN-gamma and CD107a. Most importantly, when activated ICOS- / - pmel-1 cells were adoptively tranfered into tumor-bearing mice they displayed poor proliferation and diminished antitumor activity as compared to ICOS-intact pmel-1 cells. These results provide direct evidence for the important role of ICOS in CD8+ CTL mediated antitumor immunity and have implications in the development of novel immunotherapy for cancer by manupilation of ICOS pathway.
Combined blockade of IL-4R and IKKβ inhibits melanoma growth in vivo (P2032)
Authors: Hawkins Oriana , Horton Linda , Martin Tyesha , Ayers Gregory , Richmond Ann
Published: January 2023

Constitutive activation of the nuclear factor kappa B (NF-κB) is common in melanoma, promoting tumor growth and metastasis. Therefore, targeting the NF-κB pathway is a possible therapeutic option. However, NF- κB activity is critical for the development and function of immune cells. Melanoma bearing C57BL/6 mice received BMS-345541, a selective inhibitor of the inhibitor of κB kinase (IKKβ). No difference was observed in tumor size. Tumors were analyzed by flow cytometry and intracellular cytokine staining revealed an early induction of Interleukin-4 (IL-4) within the leukocyte compartment of the tumor, with drug treatment. The production of IL-4 leads to an early switch to a tumor promoting T helper 2 (Th2) driven immune response, counteracting the anti-tumor effect of IKKβ inhibition. IL-4 receptor (IL-4R) expression on human melanoma cells has been documented; however the effects of IL-4 on these cells have generated contradictory reports. In vitro IL-4 delivery did not alter either the proliferative capacity of IL-4R expressing melanoma cell lines or their sensitivity to BMS-345541. In nude mice bearing an IL-4 transduced human A375 melanoma , BMS345541 was given in combination with IL-4R blocking antibody. This combination inhibited tumor growth compared to BMS-345541 treatment alone, similar to previous studies in C57BL/6 mice. IL-4R antagonists are in clinical trials for the treatment of asthma, making the translational prospects of this study extraordinarily viable.
Human effector memory T lymphocytes express pre-existing hypoxia-adaptation genes and are resistant to hypoxia (P2036)
Authors: Xu Yang , Metelissa Leonid , Savoldo Barbara , Dotti Gianpietro
Published: January 2023

Hypoxia that is a hallmark of solid tumors is considered an inhibitory mechanism that hampers the clinical benefits of T-cell-based immunotherapy in cancer patients. Studies have shown indeed that circulating human peripheral blood (PB) T cells activated in hypoxia have reduced proliferation and survival. However, antigen-specific T cells generated ex vivo and adoptively transferred in cancer patients are mostly (>80%) effector memory T cells (TEM) while this subset represents <20% of total circulating PB T cells. How hypoxia selectively influences TEM cells has not been studied. In sharp contrast with T cells from PB, we found that proliferation, survival and cytotoxic functions of ex vivo expanded T cells are augmented in hypoxia. Importantly, we found that TEM cells directly isolated from PB show properties similar to those of expanded T cells when compared to isolated naïve and central memory cells, suggesting that the resistance to hypoxia is an intrinsic property of TEM. Finally, resistance to hypoxia is associated with an increased hypoxia-adaptation genes. Expression of HIF-1a and downstream glycolytic genes are indeed higher in TEM cells, suggesting a genetic “pre-adaptation status” of TEM to hypoxia. Together, we show that primary TEM and ex vivo generated antigen-specific T cells are resistant to hypoxia, and this finding has relevant implication for the clinical translation of adoptive immunotherapy in cancer patients.
Nardilysin-dependent proteolysis of cell-associated VTCN1: A novel pathway and biomarker of type 1 diabetes development. (P4117)
Authors: Savinov Alexei , Radichev Ilian , Maneva-Radicheva Lilia , Amatya Christina , Parker Camille , Ellefson Jacob , Wasserfall Clive , Atkinson Mark , Burn Paul
Published: January 2023

Hyper-active T cell responses towards pancreatic β-cells highlight type 1 diabetes (T1D). V-set domain-containing T cell activation inhibitor-1 (VTCN1) protein, a B7-like negative co-stimulatory molecule, acts inhibiting T cell activation. Here, we unveil the general defect within VTCN1 pathway, common for diabetes-prone mice and subset of T1D patients. Specifically, gradual loss of VTCN1 from antigen-presenting cells and pancreatic islets progressed alongside natural T1D development and compromised VTCN1 functionality, potentiating hyper-proliferation of diabetogenic T cells. Mechanistically, loss of membrane-tethered VTCN1 relied on proteolytic cleavage mediated by the metalloproteinase nardilysin (NRD1). Cleaved soluble VTCN1 fragment (sVTCN1) was detected at high levels in peripheral blood of 47% of pediatric T1D patients, compared to 5% of healthy subjects. Blood sVTCN1 levels correlated with the disease aggressiveness and duration, highlighting potential use of sVTCN1 as the new T1D biomarker. Discovered VTCN1-NRD1 axis identifies NRD1 as a novel target for future anti-diabetic therapeutics.
Chronic inflammatory liver disease in mice expressing a CD28-specific ligand (P4099)
Authors: Corse Emily , Gottschalk Rachel , Park Joon Seok , Sepulveda Manuel , Loke P'ng , Sullivan Timothy , Johnson Linda , Allison James
Published: January 2023

Inflammation of the normally tolerant liver microenvironment precedes the development of chronic liver disease. Study of the pathogenesis of autoimmune liver diseases, such as autoimmune hepatitis (AIH), has been hampered by a lack of autochthonous chronic animal models. Through our studies of T cell costimulation, we generated transgenic mice expressing a ligand specific for the CD28 receptor, which normally shares ligands with the related inhibitory receptor CTLA-4. The mice spontaneously develop chronic inflammatory liver disease with several pathologies found in AIH, including elevated serum aminotransferases in the context of normal alkaline phosphatase and bilirubin levels, lymphocytic inflammation, focal necrosis, oval cell hyperplasia, and fibrosis. The prevalence of IFN-γ-producing CD8(+) T cells in the livers of transgenic mice suggests a role for autoimmune cytotoxicity in the chronic disease state. The CD28 ligand-specific transgenic mice will facilitate evaluation of CD8(+) T cell function in liver disease pathologies found in AIH.
Regulation of pathogenic GL7-expressing helper T cells by Tregs and CD137 (P4111)
Authors: Gauld Stephen , Leonardo Steven , De Santis Jessica
Published: January 2023

Dissecting the mechanisms that support autoantibody production is essential to our understanding of autoimmune diseases such as lupus. Recent work from our laboratory has shown that regulatory T cells (Tregs) play a key role in suppressing autoantibody production by helping to maintain B cell anergy. Our long-term goal is to understand how Tregs regulate B cell anergy. Our studies indicate that helper T cells play a key role as an intermediate between Treg and anergic B cells. Specifically, the absence of Tregs rapidly (within 4 days) drives the expansion of CD4+GL7+ helper T cells. This gross population can be further divided into 2 populations: germinal center follicular T cells (GC-TFH) and a novel non-TFH subset termed TAN cells. These GL7+ helper T cell subsets deliver potent signals to naïve and anergic B cells, inducing significant antibody production from both populations. Recent studies have begun to identify the signals that drive the expansion of GC-TFH and TAN cells in the absence of Tregs. We have shown that treatment of Treg-deficient mice with an agonistic anti-CD137 antibody reduces the expansion of CD4+GL7+ helper T cells (both GC-TFH and TAN cells). This treatment also reduced the expansion of class-switched B cells and abolished autoantibody production. Collectively, our studies indicate that CD137 signaling plays a key role in the expansion of pathogenic helper T cell subsets that support autoimmunity in the absence of Tregs.
Qa-1b has antigen presentation and immunoregulatory roles during aerogenic Mycobacterium tuberculosis infection (P3296)
Authors: Bian Yao , Shang Shaobin , Shanmuganad Sharmila , Siddiqui Sarah , Wang Chyung-Ru
Published: January 2023

The mouse MHC Ib molecule Qa-1b plays roles in both antigen presentation and immune regulation in different disease models. Further, the human homologue of Qa-1b, HLA-E, can present Mycobacterium tuberculosis (Mtb) peptides to CD8+ T cells. In viral infections, Qa-1b suppresses NK and CD8+ T cell function by interacting with inhibitory CD94/NKG2A molecules. In addition, Qa-1b can activate CD8+ T regulatory cells, which suppress CD4+ T cell responses in EAE. However, the role of Qa-1b in Mycobacterium tuberculosis (Mtb) infection is unknown. In this study, we find Qa-1b expression is upregulated in a mouse model of low-dose, aerogenic infection with Mtb. We find that Qa-1b, like HLA-E, can bind a number of Mtb peptides; a subset of these peptides are recognized by CD8+ T cells and induce IFN-γ production during aerogenic Mtb infection. To determine the overall contribution of Qa-1b to Mtb infection, we infected Qa-1b-deficient and wild-type mice. Qa-1b-deficient mice had increased production of pro-inflammatory cytokines compared to wild-type, indicating Qa-1b plays a regulatory role in Mtb infection. Infected Qa-1b-deficient mice also show increased expression of CD94/NKG2A on CD8+ T cells, indicating a potential mechanism by which Qa-1b controls immune responses in Mtb infection. Our data suggest that during Mtb infection, Qa-1b is uniquely able to both activate immune responses through antigen presentation as well as suppress immune responses via regulatory mechanisms.
T cell-intrinsic MyD88 signaling regulates Th1 polarization and IFN-γ mediated immunopathology (P3316)
Authors: Yarovinsky Felix
Published: January 2023

Activation of Toll-like receptors (TLRs) by pathogens triggers cytokine production and T cell activation, immune defense mechanisms that can cause immunopathology. Previous studies have established that Myd88-/- mice, which cannot signal through IL-1-family receptors and most TLRs, including TLR11, are acutely susceptible to infection with a protozoan parasite Toxoplasma gondii. Myd88-/- mice fail to activate T cells and produce IFN-γ essential for host resistance to the parasite. Using a comprehensive panel of cell type-specific MyD88-deficient mice, we show that T cell-intrinsic MyD88 signaling, rather than MyD88 activation in antigen-presenting cells, had a major role in Th1 polarization. T cell-specific elimination of MyD88 reduced IFN-γ secretion by CD4+ T cells. We also show that IFN-γ production by CD4+ Th1 cells during mucosal responses to T. gondii resulted in dysbiosis and the elimination of Paneth cells. Paneth cell death led to loss of antimicrobial peptides and occurred in conjunction with uncontrolled expansion of the Enterobacteriaceae family of Gram-negative bacteria. The expanded intestinal bacteria were responsible for the parasite-induced intestinal pathology. The investigation of cell type-specific factors regulating Th1 polarization during T. gondii infection identified the T cell-intrinsic TLR pathway as a major regulator of IFN-γ production in CD4+ T cells responsible for Paneth cell death, dysbiosis and intestinal immunopathology.
The inhibition of allergic airway inflammation by regulatory T cells induced with Trichinella spiralis infection. (P3322)
Authors: Kang Shin Ae , Cho Min Kyoung , Park Mi Kyung , Yu Hak Sun
Published: January 2023

In several recent reports, helminthic infections could reduce various allergic response and immune response. In previous study, we demonstrated that Trichinella spiralis infection could ameliorate allergic airway inflammation and CD4+CD25+ Foxp3+ T (Treg) cells were recruited in the lung. In order to know function of recruited T reg cell of T. spiralis infected mice, we elicited allergic airway inflammation with OVA-alum and injected Treg cell by intra-venous which was isolated from T. spiralis infected mice. Treg cells induced with T. spiralis infection inhibit OVA-induced airway inflammation. A significant reduction of immune cell infiltration and the concentrations of IL-4 and IL-13 were lower than those of control. The gene expression levels of various important homing receptors and activated Treg marker in Treg cells of each group were analyzed via real-time PCR. As a result, the expression of CTLA-4, CCR5 and CCR9 was significantly increased in Treg cells by T. spiralis infection. These data identify Treg cells induced by T. spiralis infection as capable of regulating allergic airway responses by migration to inflamed tissues and activation.
Identification of PE/PPE epitopes of Mycobacterium leprae recognized by peripheral blood mononuclear cells in leprosy (P3332)
Authors: Choi Aaron , Legaspi Anna , Hou Wanqiu , Modlin Robert
Published: January 2023

Leprosy, a chronic disease caused by the intracellular pathogen Mycobacterium leprae (M leprae), is a major health concern. Mycobacterial PE/PPE proteins may play a role in the interface of host-pathogen interaction and disease development. However, it remains unclear how the immune system recognizes those proteins and their components. Here, we have employed an Internet online-based tool called immune epitope database analysis resource (http://tools.iedb.org) to predict and analyze epitopes of M. leprae PE/PPE proteins. Thirteen candidate T cell epitopes were identified with a potential to bind a broad range of MHC class II haplotypes. Using ELISA, we found that 7/13 of predicted epitopes stimulated IFN-γ production in peripheral blood mononuclear cells (PBMCs) of tuberculoid or reversal reaction leprosy patients but not healthy donors. Among them, ML0588 (7-21) exhibited the broadest stimulatory capacity, eliciting IFN-γ production by PBMCs in 4/7 patients. Taken together, we have utilized the immune epitope database to identify seven peptides encoded by M. leprae PE/PPE proteins as strong candidate T cell epitopes for the induction of IFN-γ production in leprosy patients. These findings suggest that T cell recognition of these proteins may contribute to host defense against leprosy and be of potential use in a diagnostic test for leprosy.
The role of LIGHT a member of the TNFSF in regulation of secondary immunity to Leishmania major (P3344)
Authors: Okwor Ifeoma , Xu Giu-Lian , Uzonna Jude
Published: January 2023

We previously showed that LIGHT, (lymphotoxin like, exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes) and its receptors are critically important for initiation of CD4+ T helper (Th1) cell response and development of primary immunity to Leishmania major infection in mice. Here, we further characterized the contributions of this molecule in the activation of dendritic cells (DCs), L. major-induced IL-12 production and secondary anti-Leishmania immunity. We show that blockade of LIGHT significantly impaired DC maturation and expression of costimulatory molecules including, CD40 and CD86. In addition, blockade of LIGHT signaling lead to impairment in L. major-induced IL-12p40 production by DCs in vivo and a concomitant reduction in the number of cells recruited into the draining lymph nodes as well as early IFN-γ production by CD4+ T cells following L. major infection. Furthermore, we show that LIGHT is required at both priming and maintenance stages of anti-Leishmania immunity. Interestingly, blockade of LIGHT had no effect on delayed-type hypersensitivity (DTH) response and control of parasite burden following secondary L. major challenge. Collectively, these results show that LIGHT is critical for IL-12 production by DCs and subsequent priming and maintenance of IFN-γ response by CD4+ T cells during primary but is dispensable during secondary anti-Leishmania immunity.
Production of monoclonal camelid antibodies by means of hybridoma technology (P3376)
Authors: Holzloehner Pamela , Schliebs Erik , Maier Natalia , Füner Jonas , Micheel Burkhard , Heilmann Katja
Published: January 2023

Here we describe the first monoclonal immunoglobulins produced by camelid hybridomas. Hybridomas were generated by using the CBF7 heteromyeloma line to fuse peripheral blood lymphocytes from New World camels (llamas, alpacas) and Old World camels (Bactrian camels). The hybridomas produced immunoglobulins of IgG1, IgG2 and IgG3 subclasses as defined by both enzyme immunoassays and PCR and subsequent gene sequencing. The subclass-specific murine monoclonal antibodies were also produced in the experiments described here. They could be used to detect conventional four-chain camelid antibodies (of subclass IgG1) and heavy chain-only antibodies (both of subclasses IgG2 and IgG3). The different monoclonal heavy chain-only immunoglobulins and the producing hybridomas are of special interest since they can be used to produce further secondary reagents (murine monoclonal antibodies) for the characterization of different camelid antibodies and to solve some as yet unresolved problems in the molecular biology of heavy chain antibody-producing cells. Most importantly, the technique described here can be the basis for the production of full-length heavy chain-only monoclonal antibodies of camelids directed against specific antigens.
Multiplex gene expression analysis for low abundance intracellular mRNAs in individual cells by RNA flow cytometry (P3381)
Authors: Park Emily , Lomas Woodraw , Hanley Mary , Mittar Dev , Su Nan , Luo Yuling , Maino Vernon
Published: January 2023

A variety of RNA analysis technologies is available for the detection of RNA transcripts from bulk cell populations. However, the techniques for RNA detection from single cells have been limited due to lack of sensitivity in detecting specific mRNAs present at low abundance in individual cells. By designing novel target-specific probes and adapting an in situ signal amplification method based on the RNAscope® detection platform, we demonstrate the specific and sensitive detection of intracellular RNAs in single cells by flow cytometry (RNAFlow). This method had sufficient sensitivity to distinguish cells containing low abundance RNA transcript. Furthermore, multiple distinct RNA targets were simultaneously detected with a high specificity in single cells without interference. The method can quantify the frequency of cells expressing specific RNA as well as the number of RNA copies in each expression-positive cell. We will present quantitative data to characterize RNA target-specific signal detection, demonstrated in applications such as HIV infection and immune activation. The RNAFlow assay, independently or as combined assay with co-protein detection, represents a valuable research tool for the specific and sensitive detection of multiple RNA transcripts in individual cells in heterogeneous biological specimens. Overall, this method will be useful for the analysis of functionally important RNA species from single cells, even at very low copy numbers.
Simplified T & B lymphocyte enumeration on a novel cell analyzer (P3390)
Authors: Clor Julie , Gillis Katherine , Tyagarajan Kamala
Published: January 2023

Characterization and enumeration of T and B cell lymphocyte sub-populations is crucial in immunological research applications for understanding of immune status and response. This analysis has typically been performed using sophisticated single platform flow cytometry or double platforms, to which many researchers may have limited access. In this study, we evaluated performance of a novel, low-cost, small footprint platform, the Muse™ Cell Analyzer which is based on microcapillary cytometry and utilizes guided touchscreens, for immunophenotyping analysis. Whole blood and peripheral mononuclear blood cell samples a were analyzed using simplified, no wash, novel two color strategies and results for CD4 T cells, CD8 T Cells and B cells count and percentage of lymphocytes were compared against well-established predicate methods. Comparison of results demonstrates good correlations and low bias for each strategy. Precise results were obtained for all assays tested as shown by the low coefficient of variation (CV<10%). Low operator to operator variability with inter-operator CV’s of <10% was also observed for the analysis. Further, T or B lymphocyte characterization on samples is performed using low number of cells. The accurate and precise enumeration and evaluation of T and B lymphocytes in a simple, accessible system can empower researchers to obtain information on critical T& B lymphocyte populations of interest with ease.
Interleukin-4 directly promotes intestinal mast cells expansion in the context of food allergy (P3256)
Authors: Burton Oliver , Darling Alanna , Zhou Joseph , Noval-Rivas Magali , Jones Tatiana , Gurish Michael , Chatila Talal , Oettgen Hans
Published: January 2023

Interleukin-4 is a pivotal cytokine in the development of allergic disorders. Mast cells are key effectors of food allergen-driven anaphylaxis, and IL-4 has been implicated in their function. More recently, however, we and others have shown that IL-4 has a mix of inhibitory and stimulatory effects on bone marrow-derived mast cells. We therefore sought to clarify the direct effects of IL-4 on intestinal mast cell function and growth. In this study, we demonstrate that IL-4 drives a marked intestinal mastocytosis in mice. Similar effects were observed in animals expressing Il4rαF709, an activating variant of the IL-4 receptor α-chain that confers susceptibility to food allergen-driven anaphylaxis. Il4rαF709 mice show heightened sensitivity to IgE-mediated anaphylaxis without increased sensitivity to mast cell mediators. Exposure to IL-4 upregulates anti-apoptotic Bcl proteins in intestinal mast cells, leading to enhanced survival and proliferation via STAT6. These effects are intensified by the Il4rαF709 signaling variant. Mixed bone marrow chimera experiments demonstrate that Il4rαF709 mast cells hold a substantial competitive advantage over wild-type cells in populating the intestine in response to food allergen stimulation. IL-4Rα-deficient mast cells do not effectively expand, establishing a cell-intrinsic effect of IL-4 in intestinal mast cell homeostasis. Our results demonstrate that IL-4-signaling is a key determinant of mast cell expansion in food allergy.
Use of in vivo caspase inhibitor in rat burn and infection injury prevents gut bacterial translocation and modulates CD4+ T cell effector immune response. (P3272)
Authors: Fazal Nadeem
Published: January 2023

Our previous studies have focused on gut-associated lymphoid tissues, failure of intestinal gut barrier, bacterial translocation, and sepsis exploring the roles of antigen-presenting cells, DCs, CD4+ T cell and regulatory T cells in immunosuppression following acute burn and/or burn-plus-infection injury. In an established rodent model of burn plus infection we tested in vivo use of caspase inhibitor and studied T cell effector immune response and gut barrier function. Z-VAD-FMK, a highly specific, cell-permeable, and irreversible inhibitor of caspases was given to rats and were then assessed for mortality, bacterial translocation, T cell receptor expression and apoptosis. The results suggested an increase in CD4+CD25+ T cell phenotype in splenocytes of burn as compared to sham rats, however such an elevation in CD4+CD25+ expression was reduced in CLP and CLP+Burn+Caspase-Inhibitor groups. The results also showed that there was an increase in adhesion receptors (LFA) and decrease in CD62L in burn, whereas no difference in these adhesion receptor expressions was ascertained in Burn+CLP and Burn+CLP+Caspase-Inhibitor treated rats. A decrease in homing receptor (CD49d) was also noticed in Burn+CLP and Burn+CLP+Caspase-Inhibitor-treated animals. The Colony Forming Unit (CFU) results showed absence of bacterial translocation in rats that were subjected to burn+CLP+Caspase-Inhibitor, however there was significant bacterial translocation in rats from the group of CLP and burn only.
Allergen-induced lysosomal CCL20 release from bronchial epithelial cells (P3284)
Authors: Lajoie Stephane , Webb Mark , Wills-Karp Marsha
Published: January 2023

Although allergic asthma is a chronic inflammatory disease of the lung mediated by Th2 cells, the pathways leading to this response are not clear. Recent studies have highlighted influence of pulmonary dendritic cells (DCs) on T-cell differentiation. Rapid DC recruitment into the bronchial mucosa has been well documented in animals and humans in response to allergen. Central to early DC recruitment, CCL20 is the only known ligand for CCR6, a receptor preferentially expressed on immature DCs. The potential importance of CCL20/CCR6 in allergic sensitization was demonstrated in a cockroach model of allergic asthma in which Ccr6-/- mice were protected against asthma. At present, the mechanisms by which allergen induces secretion of DC-attractant chemokines are unknown. We previously showed that bronchial epithelial cells release CCL20 relatively rapidly after house dust mite (HDM) (Nathan, 2009). This induction was not prevented by the inhibiting protein synthesis (cycloheximide) suggesting release through a transcriptionally independent mechanism. These data suggested that HDM may induced the release of preformed stores of CCL20
Glucocorticoid-induced TNF receptor family related protein ligand regulates the egress of monocytes from the spleen (P3288)
Authors: Liao Gongxian , van Driel Boaz , Magelky Erica , O’Keeffe Michael , Herzog Roland , Mizoguchi Emiko , Bhan Atul , Terhorst Cox
Published: January 2023

Glucocorticoid-Induced TNF Receptor family-related protein Ligand (GITR-L) is expressed on antigen presenting cells. Surprisingly, GITR-L−/−Rag−/− mice develop a markedly milder colitis than Rag−/− mice upon transferring CD4+CD45RBhi T cells or by administering agonistic anti-CD40 antibody. In both colitis models as well as in a peritonitis model a reduced number of Ly6C+CD11b+MHCII+ macrophages was found in the inflamed sites of GITR-L−/− mice. By contrast, the number of non-differentiated monocytes was approximately three-fold higher in the spleen of inflamed GITR-L−/−Rag−/− mice than in Rag−/− mice. The formation of an active dimer of the angiotensin II type 1 receptor (AT-1) by splenic monocytes, which regulates their egress, was dynamically affected by the GITR-L deficiency. Infusion of Angiotensin II caused retention of splenic monocytes in GITR-L−/− but not wt mice. We conclude that upon induction of inflammation GITR-L regulates egress of monocytes from the spleen, thus impacting intestinal homeostasis in mice.
Resveratrol and its derivatives modulates TH17 differentiation and attenuates experimental autoimmune encephalitis (P5139)
Authors: Yen Hung-Rong , Chung Pei-Chen , Yang Hsu-Ling
Published: January 2023

The pathogenic role of IL-17 and TH17 cells in inflammation and autoimmunity has prompted the development of new inhibitors by pharmacologic approaches. Here we demonstrated resveratrol (3,5,4'-trihydroxy-trans-stilbene) and its derivatives from natural products and traditional Chinese medicine, inhibit TH17 differentiation in a concentration-dependent manner in vitro. CD4 T cells polarized in the TH17 condition with resveratrol expressed a relatively lower level of IL-17, ROR-gt, and TH17-related gene expression compared to the non-treated cells. To further determine its efficacy in vivo, we administered candidate drugs in a murine model of experimental autoimmune encephalitis, resveratrol and its derivatives ameliorates the disease severity and the production of TH17 in vivo. Our preliminary data fit the niche that is required to modulate TH17 for immunotherapy and we suggest that, to treat autoimmune disease, administering resveratrol and its derivative could be a promising pharmacologic approach to achieve the goal. Additional mechanisms are being explored.
Resveratrol ameliorates lupus by epigenetic modulation of Foxp3 and IL-17 genes resulting in differential regulation of regulatory T cells and Th17 cells via activation of aryl hydrocarbon receptor (P5129)
Authors: Singh Narendra , Singh Udai , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Resveratrol (RES), a polyphenolic compound found in plant products, possesses anti-inflammatory, antioxidant, and anticancer properties. We investigated the use of RES for the treatment of lupus using an animal model. RES treatment significantly suppressed Pristane-induced lupus in SJL/J mice. There was significant downregulation of IL-17 but upregulation of FoxP3 expression. RES initiates its early signaling via AhR and recent studies have suggested that AhR may regulate T cell differentiation. We examined the effect of RES on differentiation of Tregs and Th17 cells, microRNA (miR) profile in T cells, and epigenetic modulation of Foxp3 and IL-17 genes. There was a significant difference in the miR profiles in T cells of RES-treated mice, when compared to vehicle-treated mice. Analysis of 609 miRs revealed dysregulation of 112 miRs including downregulation of miRs (miR-31,-490, -217, and -190) that regulate FoxP3 and upregulation of miRs (miR-200, -203, -431, -494, and -495) that regulate IL-17. Upon examination of epigenetic effects of RES on Foxp3 and IL-17 promoters, we observed significant demethylation of Foxp3 promoter but methylation of IL-17 promoter. Culture of T cells in the presence of RES led to significant increase in Tregs but decrease in Th17 cells in vitro and in vivo. Together, these data demonstrated that RES via AhR regulates epigenetic modulation and can have differential effects on T cell differentiation into Tregs/Th17 cells causing suppression of lupus.
Critical role of all-trans retinoic acid in stabilizing human nTregs under inflammatory conditions (P5133)
Authors: Zheng Song Guo , Wang Julie , Horwitz David , Quismorio Francisco , Lu Ling
Published: January 2023

Recent studies have demonstrated that thymus-derived naturally-occurring CD4+Foxp3+ regulatory T cells (nTregs) in both human and mouse are unstable and dysfunctional in the presence of pro-inflammatory cytokines. All-trans Retinoic Acid (atRA), the active derivative of vitamin A, has been shown to regulate Treg and T effector cell differentiation. Herein, we report that atRA prevents human nTregs from converting to Th1 and/or Th17 cells and sustains their Foxp3 expression and suppressive function in vitro or in vivo following encountering with IL-1 and IL-6. Interestingly, adoptive transfer of human nTregs pre-treated with atRA significantly enhanced their suppressive effect on xeno-graft versus host diseases (xGVHD) and only these cells after stimulating with IL-1/IL-6 sustained the functional activity against xGVHD in a humanized animal model. atRA suppresses IL-1R upregulation and accelerates IL-6R downregulation and diminishes their signaling events, as well as increases histone acetylation on FOXP3 gene promoter and CpG demethylation in CNS regions of FOXP3 gene locus. These results strongly implicate that nTregs primed with atRA may represent a novel treatment strategy to control established chronic immune-mediated inflammatory diseases.
Investigating TLR4-signaling mechanisms in CHME-5 human microglial cells and the effects of β-funaltrexamine treatment (P1236)
Authors: Figueroa Leandra , Das Subhas , Buck Daniel , Stevens Craig , Davis Randall
Published: January 2023

Neuroinflammation is instrumental in neuronal damage observed in various neuropathologies. Thus, understanding pro-inflammatory pathways of human glial cells is important in identifying potential pharmacological strategies to modulate inflammatory signaling in these cells. Microglia-derived cytokines and chemokines are instrumental in neuroinflammation and several proinflammatory mediators activate these cells to include bacterial lipopolysaccharide (LPS) that signals through toll-like receptor 4 (TLR4). Microglial TLR4 signaling is not well studied; therefore, our goal is to investigate the molecular mechanisms of TLR4 microglial signaling, which entailed treatment of human microglial cells (CHME-5) with LPS. LPS induced NF-κB activation, demonstrated by increased NF-κB p65 binding activity and phosphorylated p65 expression in CHME-5 cells. TLR4 expression also increased alongside NF-κB activation. On the other hand, co-exposure to the μ-opioid receptor antagonist, β-funaltrexamine (β-FNA), down-regulated LPS-induced NF-κB activation. To date, our data suggest that the inflammatory actions of microglial TLR4 signaling may be partly due to modulation of NF-κB activation. Thus, we have also initiated studies to elucidate the effects of LPS and β-FNA on other key TLR4 signal transduction proteins. Together, these ongoing studies are expected to further elucidate the mechanisms of LPS-induced TLR4 signaling in human microglia.
Herpes simplex virus type 2 induces innate immune response via TLR4-MyD88/Mal-NF-kB axis in human cervical epithelial cells (P1220)
Authors: Li Hui , Liu Hongya , Chen Kai , Feng Wenqiang , Wu Xinxing , Liu Xuefeng
Published: January 2023

Toll-like receptors (TLRs) play a crucial role in regulating innate immune response to HSV-2 infection. Current studies on TLRs and viral infection have used either immuno-competent cells and/or murine systems. The innate immune pathway in response to this virus in its natural host cells remains unclear. We have established an in vitro HSV-2 acute infection model with Human cervical epithelial (HCE cells, the primary target and natural host cells for HSV-2) to investigate the role of TLRs-mediated innate immune response to HSV-2. In current study, we found that HSV-2 infection induced NF-kB reporter activity and expression of cytokines are TLR4-dependent. Knockdown experiments demonstrated that the adaptor molecules MyD88 and Mal of the TLRs signaling pathways are required in the HSV-2 induced TLR4-dependent NF-kB activation in HCE cells. Western blot assay suggested that knockdown of TLR4 decreased the phosphorylation of IRAK1 and inhibitor of NF-kB (IkB-a) upon HSV-2 infection. Finally, decreased expression of either TLR4 or MyD88/Mal alone or both significantly abolished productions of IL-6 and IFN-beta by ELISA analysis. Taken together, our results reveal for the first time that there exists the pathway via TLR4-Mal/MyD88-IRAK1-NF-kB axis in response to HSV-2 infection in its natural host cells.
MeCP2 enforces Foxp3 expression to determine natural regulatory T cells’ resilience to inflammation (P1023)
Authors: Li Chaoran , Jiang Shan , Sevilla Jose , Li Qi-Jing
Published: January 2023

Foxp3+ regulatory T cells are crucial for peripheral tolerance and immune homeostasis. During inflammation, the steady expression of Foxp3 in mature regulatory T cells is essential for maintenance of their lineage identity and suppressive function. However, the molecular machinery governing regulatory T cells’ resilience to inflammation induced Foxp3 destabilization is not fully understood. Here, we demonstrate that Methyl-CpG binding protein 2, an eminent epigenetic regulator known primarily as the etiological factor of the neurological disorder Rett syndrome, is essential to sustain regulatory T cells’ Foxp3 expression during inflammation. In response to inflammatory stimuli, Methyl-CpG binding protein 2 is specifically recruited to the CNS2 region of the foxp3 locus, where it collaborates with CAMP responsive element binding protein 1 to promote local histone H3 acetylation, thereby counteracting inflammation induced epigenetic silencing of foxp3. Consequently, regulatory T cell specific deletion of Methyl-CpG binding protein 2 is sufficient to induce spontaneous immune activation in young adult mice. Furthermore, we demonstrate that Foxp3 expression diminishes with time in Methyl-CpG binding protein 2 deficient regulatory T cells, resulting in their failure to suppress Th17-mediated colitis. Thus, Methyl-CpG binding protein 2 serves as a critical safeguard that confers regulatory T cells with resilience against inflammation.
The induction of anterior chamber associated immune deviation with Type II collagen (P1045)
Authors: Ashour Hossam , Farooq Shukkur
Published: January 2023

The administration of antigens into the anterior chamber (AC) of the eye induces a special form of antigen-specific peripheral immune tolerance termed AC-associated immune deviation (ACAID), which prevents delayed-type hypersensitivity (DTH) responses and other inflammatory responses. Type-II collagen (CII) is highly expressed in cartilage tissues and has been linked to Rheumatoid arthritis, aging, and osteoarthritis. To explore the potential for ACAID induction via CII, we checked for different signs of ACAID generation following the AC injection of CII in BALB/c mice. We hypothesized that the mechanism of ACAID induction involves efferent T regulatory cells (Tregs). Both local adoptive transfer (LAT) assays and DTH assays were performed. Results indicated that ACAID induction was driven by the AC injection of CII. Spleen cells of mice injected with CII in the AC significantly suppressed DTH responses. ACAID induction was mediated by efferent Tregs in the spleen. CII-mediated ACAID induction could have multiple therapeutic implications, especially in autoimmunity.
Establishing the molecular size threshold of immune synapse penetration: Implications for drug access (P4263)
Authors: Cartwright Adam , Griggs Jeremy , Davis Daniel
Published: January 2023

Natural Killer (NK) cells survey target cells through activating, inhibitory and adhesion receptors that bind their ligands at the immune synapse. This dense mesh of protein-protein interactions at the intercellular contact may create a seal, or gasket, around the synapse to prevent the leakage of secreted proteins, but this remains poorly understood. Nanometer scale fluorescent dextrans of known sizes were used to determine the size threshold of molecules that could penetrate the immunological synapse between NK and target cells, by measuring fluorescence accumulation within the intercellular region. Dextran was completely excluded from the synapse when its hydrodynamic diameter was above 13nm, whereas a 4nm dextran was able to diffuse in and out of the synapse. This showed that the synapse represents a physical gasket through which molecules can penetrate in a size-dependent manner. This establishes a size threshold of molecules that are able to enter the synapse. Drugs which target proteins within the synapse should be engineered to these dimensions in order to penetrate the synapse.
Topical CpG adjuvant enhances immune response to subcutaneous antigen by modulating the cutaneous lymph node environment (P4267)
Authors: Cheng Wing-Ki (Vicki) , Plumb Adam , Wee Kathleen , Kollmann Tobias , Abraham Ninan , Dutz Jan
Published: January 2023

Current vaccines are mainly administered by intramuscular route but the skin harbors many immune cells and can be used as a site of immunization. Topical CpG oligodeoxynucleotide (ODN), a Toll-like receptor 9 (TLR9) agonist, improves humoral and cell-mediated immune responses to locally injected protein-based vaccines in mice but the mechanisms are still unclear. Topical CpG ODN is internalized by CD11c+ cells that are detected within the skin draining lymph nodes (SLN) at 48 hours post treatment. Ablation of TLR9 in the hematopoietic compartment of bone marrow chimeric mice abrogates antigen-specific CD8+ T cells production. TLR9 ablation in the stromal compartment also decreases topical CpG adjuvant effect. Topical CpG ODN differentially modulates the environment of SLN compared to subcutaneous administration within 24 hours of treatment (with 4-7 fold increased expression of 4 genes: Ccl4, Cxcl3, Ifng and Il11). A higher proportion of antigen-specific CD4+ T cells in the SLN express tissue-homing molecules (P- and E-selectin ligand) when adjuvant is administered topically compared to subcutaneously. Egress of lymphocytes from SLN is necessary for optimal CpG adjuvant effect using a contact hypersensitivity model. We propose that topical CpG ODN activates stromal cells as well as hematopoietic-derived cells in the skin, to subsequently modulate the environment of SLN. These changes enhance protective humoral and cell-mediated immune responses.