bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct interaction of recombinant TAPBPR with MHC-I molecules: stabilization of peptide-free MHC-I promotes high affinity peptide loading (APP5P.102)
Authors: Morozov Giora , Zhao Huaying , Mage Michael , Boyd Lisa , Venna Ramesh , Norcross Michael , McMurtrey Curtis , Hildebrand William , Schuck Peter , Jiang Jiansheng , Natarajan Kannan , Margulies David
Published: January 2023

The loading of MHC-I molecules with peptides for cell surface display is a crucial step in self-tolerance and activation of CD8 T cells. We studied TAPBPR (TAP binding protein-related) protein, a tapasin homolog, which is widely expressed and IFN-γ inducible, but is not part of the classical MHC-I peptide-loading complex. We produced recombinant soluble TAPBPR and evaluated its interactions with several recombinant MHC-I molecules in vitro, by gel-shift, size exclusion chromatography, ultracentrifugation, and surface plasmon resonance. We show that TAPBPR binds MHC-I after photolysis of a bound peptide, and that the TAPBPR/MHC-I complex is dissociated by exposure to peptides that bind the MHC-I molecule, indicating a role of TAPBPR in stabilizing a peptide-receptive form of the MHC-I/β2m complex. Peptide-dependent release of MHC-I from TAPBPR is directly proportional to the peptide’s affinity for MHC-I. Peptide binding experiments indicate a role for TAPBPR in selection of high affinity peptides. Mutagenesis of TAPBPR and MHC-I confirm the importance of amino acid residues conserved with the putative tapasin/MHC-I binding site and reveal additional residues important for the TAPBPR/MHC-I interaction. Molecular docking simulations suggest a detailed mechanism for the interaction of TAPBPR with peptide free MHC-I. These studies are consistent with the view that TAPBPR functions as a chaperone that stabilizes peptide-free MHC-I to permit binding of high affinity peptides.
ERAP1-ERAP2 dimers trim MHC I-bound precursor peptides: a molecular understanding of peptide editing (APP5P.103)
Authors: Bouvier Marlene , Chen Hanna , Li Lenong , Weimershaus Mirjana , Evnouchidou Irini , Van Endert Peter
Published: January 2023

The human endoplasmic reticulum aminopeptidase 1 (ERAP1) and ERAP2 are critically important in the final processing of MHC class I antigens. To date, the molecular context of peptide trimming by ERAPs, and how ERAPs shape antigen repertoires, remain open questions. Using a cell-free system composed of ERAP1 and ERAP2 heterodimers (ERAP1/2), MHC class I molecules, and N-terminally extended model and natural peptides, we characterized the function of ERAPs. We provide conclusive evidence that ERAP1/2 trims MHC I-bound precursor peptides to the final lengths, albeit more slowly than the corresponding free precursors. We show that trimming of MHC I-bound precursors by ERAP1/2 increases the conformational stability of MHC I/peptide complexes. We will discuss a molecular mechanistic model of peptide editing by ERAP1/2 that unifies and advances our understanding of these important enzymes.
Identification of self-specific CD4+ T cell subsets resistant to PD-1 blockade (BA11P.130)
Authors: Martinov Tijana , Pauken Kristen , Nelson Christine , Spanier Justin , Heffernan James , Sahli Nathanael , Osum Kevin , Jenkins Marc , Vezys Vaiva , Fife Brian
Published: January 2023

The inhibitory receptor programmed death (PD)-1 and its ligand PD-L1 regulate T cell function to limit autoimmunity. PD-1 is highly expressed on exhausted T cells, limiting their antiviral or antitumor activity. Even though PD-1 pathway blockade has gained momentum in cancer treatment, it is unclear how PD-1/PD-L1 blockade impacts different T cell subsets. We examined the effects of PD-1 blockade on self-reactive CD4+ T cells in mouse models of varying autoimmune susceptibilities. We used insulin-peptide/MHC Class II tetramers to track endogenous insulin-specific CD4+ T cells in diabetes-prone (NOD), and diabetes-resistant (NOR and B6.g7) mice. PD-1 was expressed at lower levels on insulin-specific cells in diabetes-resistant mice compared to cells from NOD mice. Moreover, PD-1 pathway blockade accelerated autoimmunity in all NOD mice and 30% of NOR mice, but B6.g7 mice remained protected from disease. By transferring congenically marked islet-specific CD4+ T cells into pre-diabetic NOD mice, we asked whether anti-PD-L1 reinvigorates anergic cells. Surprisingly, anergic cells remained functionally blunted compared to effector cells after anti-PD-L1, suggesting that established anergic cells were not dependent on PD-1 signaling to maintain their unresponsive state. This work highlights how the differentiation status of a T cell may predetermine its susceptibility to PD-1 blockade, and influence autoimmunity or antitumor responses that ensue.
The dynamics of T cell restimulation in the pancreatic islets indicate a period of T cell tolerance during autoimmune diabetes (BA11P.125)
Authors: Friedman Rachel , Lindsay Robin , Gebert Matthew , Bradley Brenda , Krummel Matthew , Haskins Kathryn , Jacobelli Jordan
Published: January 2023

In type 1 diabetes, the pancreatic islets are an important site for therapeutic intervention since immune infiltration and attack of the islets is well established at diagnosis. To understand how T cell stimulation and function evolve at different stages of islet infiltration, we initially analyzed the dynamics of T cell movement and T cell interactions with CD11c+ APCs within individual islets. Using 2-photon imaging, we defined a correlation between increased islet infiltration and increased T cell motility. Early T cell arrest was antigen-dependent and due, at least in part, to antigen recognition through stable interactions with CD11c+ APCs in the islets, which were lost with increased islet infiltration. Stable T cell-APC interactions resulted in TCR clustering, supported IFN-γ production, and CD8 T cell killing of islet APCs. As islet infiltration increased, T cell motility became antigen-independent, with a loss of sustained interactions with APCs resulting in reduced IFN-γ production and APC killing. These studies suggest that the autoimmune T cell response in the islets is temporarily dampened during the course of islet infiltration and disease progression. We suggest that an infiltrating population of CD11c+ APCs, which we can distinguish from islet-resident APCs via expression levels of CX3CR1, plays a role in establishing a period of T cell tolerance within the pancreatic islets following initial T cell infiltration.
Influence of cytokines and TLR agonists on CD49a, CD49b and CD103 expression by T cells (CAM4P.152)
Authors: Melssen Marit , Hutchison Ciara , Bullock Timothy , Slingluff Craig
Published: January 2023

Integrins CD49a (alpha1), CD49b (alpha2) and CD103 (alphaE) are highly expressed on tumor infiltrating lymphocytes (TILs) compared to circulating lymphocytes. Interactions of the integrins with known ligands support retention of lymphocytes in peripheral tissues; thus, they may be considered retention integrins (RI). T cells in peripheral tissues expressing RI have a cytotoxic phenotype and RI+ TIL are associated with improved patient survival. Despite their importance in retention of tumor reactive TILs, little is known about pathways inducing RI expression, except that TGFβ induces CD103. We hypothesized that other cytokines, TLR agonists, and T cell receptor (TCR) stimulation can induce RI expression by circulating T cells. We stimulated PBMC with cytokines or TLR agonists after TCR stimulation, and evaluated RI expression by flow cytometry after 2-7 days. Both CD103 and CD49a are induced by TGFβ, most strikingly on activated T cells. IL2 and IL10 also influence CD49a expression. CD49b expression is increased by IL2, IL4 and IL10, although this depends on T cell subset and activation state. TLR2, TLR6 and TLR7 agonists also appear to induce CD49a and CD49b. In conclusion, our results support our hypothesis by showing that RI expression is tightly regulated by cytokine and TCR stimulation. Furthermore these regulating factors have different effects among the RI, suggesting a different role for each.
A new technology for in vitro chemotaxis assays (CAM4P.159)
Authors: APPLEDORN DANIEL , O'Clair Lindy , Tilhonenko Maria , Roddy Meagan , Schroeder Kirk
Published: January 2023

Chemotactic-driven cell migration is an important biological process in many areas of life science research including immunology, cancer and developmental biology. The dominant high-throughput (96-well format) solution for measuring cellular chemotaxis in vitro is the Boyden-chamber. However, Boyden chambers have some inherent liabilities including: a) the inability to directly visualize the cell migration process, b) a requirement of tens of thousands of cells per well, c) the need to label cells for quantitation, d) a relative insensitivity to surface integrin signaling, and e) high random migration artifacts. Microfluidic solutions have sought to eliminate some of these deficiencies, specifically by providing the ability to visualize the cell migration process, but at the expense of relatively low throughput and cumbersome quantitation methods. We have developed a new technology which eliminates most of the deficiencies of existing Boyden chambers and most importantly, provides a direct visualization of the migration process as it occurs. Integrated metrics precisely quantify the chemotactic response using significantly fewer cells (500-5000 cells per well) in a 96-well format. This assay method does not require labeling for quantitation, has a kinetic read-out, and is sensitive to surface-integrin signaling. Pharmacological data examples from T cells, neutrophils, cancer cells, and endothelial cells will be shown with observations enabled by the real-time visualization.
Sphingosine 1-phosphate receptor-3 contributes to host defense against polymicrobial sepsis via modulating macrophage chemotaxis (CAM4P.161)
Authors: Shu Qiang , Hou JinChao , Chen QiXing , Fang XiangMing
Published: January 2023

Objective Sphingosine 1-phosphate receptor-3 (S1P3) is an important G protein-coupled receptor regulating host immune response. However, the role of S1P3 in polymicrobial sepsis remains unclear. Method Sepsis was induced in wild-type (WT) and S1PR3-deficient mice via cecal ligation and puncture (CLP). The mortality of mice, as well as organ injury and bacterial clearance during sepsis were assessed. The role of S1PR3 in sepsis was further confirmed by administration of an agonist peptide representing part of the intracellular domain of S1PR3 (GPS-725.017) in septic WT mice. Results S1PR3 deficient mice showed reduced survival rate, increased lung and liver damage, and more bacterial burden during sepsis. There was also significantly less macrophages chemotactic into the peritoneal cavity in the S1PR3 deficient mice. GPS-725.017 could active S1PR3, including internalization of the membrane protein S1PR3 into cytoplasm and activation of MAPKs signaling. In vivo, GPS-725.017 treatment increased recruitment of phagocyte into the peritoneal cavity, enhanced bacterial clearance and improved survival during sepsis. Conclusion The present findings uncover S1PR3 might be a critical modulator of macrophage chemotaxis in the pathogenesis of sepsis. The detailed mechanisms need to be further addressed.
Myosin-IIA regulates B cell leukemia migration and bone marrow engraftment and its inhibition reduces leukemia dissemination and prolongs survival (CAM4P.148)
Authors: Jacobelli Jordan , Wigton Eric , Thompson Scott
Published: January 2023

Leukemia relapses often affect peripheral organs such as the central nervous system (CNS), which acts as a sanctuary site for leukemia cells to escape anti-cancer treatments. Leukemia dissemination requires migration of cells from the blood circulation into target tissues by extravasation, similar to normal leukocyte migration. To extravasate, leukemia cells cross through vascular endothelial walls via a process called trans-endothelial migration (TEM), which requires cytoskeletal remodeling. However, the specific cytoskeletal effectors of leukemia extravasation are not fully known. We examined the role of Myosin-IIA (MyoIIA), a class II myosin motor protein, in leukemia cell extravasation and if its inhibition would hinder leukemia dissemination into the CNS. We inhibited MyoIIA expression using shRNA in a mouse model of Bcr-Abl driven B cell acute lymphoblastic leukemia (B-ALL). MyoIIA depletion did not affect apoptosis or the growth rate of B-ALL cells. Instead, MyoIIA was required for B-ALL cell chemotaxis towards CXCL12 and for completion of TEM. Inhibition of MyoIIA also reduced the ability of B-ALL cells to efficiently engraft and disseminate in vivo, including a highly significant reduction in CNS infiltration. Finally, MyoIIA inhibition prolonged survival in an in vivo leukemia transfer model. Our data implicate MyoIIA as a key regulator of leukemia cell migration making it a promising target to inhibit leukemia dissemination in vivo and reduce relapse frequency.
Tracking of CD8+ effector T cells in intact lung: analysis of environmental guidance (CAM4P.151)
Authors: Mrass Paulus , Ellenwood Sean , Moses Melanie , Cannon Judy
Published: January 2023

Effector T lymphocytes patrolling peripheral tissues protect organisms against infection and cancer. Recent two-photon imaging studies have revealed that T cells move with high speed through the interstitium of inflammatory tissues. We are interested in assessing migration strategies of tissue-infiltrating T cells and determining how T cell motility affects the efficiency of target search. We perform two-photon imaging using a murine endotoxin lung model to define the rules that govern motility of CD8+ effector T cells. We track individual T cells moving in intact lung for up to three hours but we do not find any evidence for long-range guidance cues for T cells, consistent with observations in other environments. Nevertheless, computational analysis of trajectories with an automated image-analysis algorithm reveals a significant alignment of T cells moving in conjunction with lung-associated vessels. Our results suggest that the architecture of the lung actively guides the migration of effector T cells and may act to limit their movement within tissues. Long term tracking of T cells moving in live tissue can provide a better understanding of the underlying mechanisms driving the search-efficiency of T cells during the effector phase of an immune response.
The TNF-family cytokine receptor death-receptor 3 (TNFRSF25) is required for nephritis but not systemic autoimmunity in the lpr murine model of lupus (CCR5P.209)
Authors: Hayes Erika , Villarreal Sarah , Ferdinand John , Engel Odile , Meylan Francoise , Siegel Richard
Published: January 2023

DR3 (Tnfrsf25) is a death domain-containing tumor necrosis factor receptor found primarily on activated T cells and innate lymphocytes. Interaction with its ligand, the TNF-family cytokine TL1A, plays a critical role in diverse autoimmune disease models in facilitating T cell accumulation at the site of inflammation while not affecting the systemic response. To determine whether DR3 could play a role in systemic autoimmunity, we studied the role of DR3 in the MRL/lpr mouse model of lupus. Lpr mice have a mutation in the TNF family receptor Fas (CD95, Tnfrsf6) that impairs its expression, causing systemic autoimmune disease that mimics systemic lupus erythematosus (SLE) observed in humans. DR3 deficient lpr mice developed identical degrees of lymphadenopathy, splenomegaly and double-negative T cells compared to control littermates. Expansion of effector T and B cell subsets were also not dependent on DR3. However, DR3 deficiency significantly protected MRL/lpr mice from IgG and C3 glomerular immune complex deposition and DR3 deficient kidneys had decreased medullary T cell accumulation, decreased periglomerular and medullary macrophage infiltrates and reduced proteinuria. These findings show that DR3 specifically promotes renal pathology in the MRL/lpr mouse model of lupus nephritis without affecting systemic autoimmunity or other features of Fas deficiency. Blockade of TL1A-DR3 interactions may be a novel strategy to counter nephritis and other organ damage in human SLE.
Study of the biological activities of a novel monoclonal anti-mouse CX3CR1 antibody, clone SA011F11. (CCR5P.213)
Authors: Lin Leo , Soto Hortensia , Oida Takatoku , Yang Xifeng , Moyron-Quiroz Juan
Published: January 2023

CX3C chemokine receptor 1 (CX3CR1) is a 7 transmembrane G-protein coupled receptor involved in the recruitment of cells to inflamed sites, cell adhesion, and cell extravasation; its ligand is CX3CL1. It has been reported that CX3CR1 is expressed on different cell types such as resident and alternatively activated macrophages, microglia, subsets of monocytes, DCs, NK cells, memory T cells, B cells, mast cells, astrocytes, and some tumor cells. Here we describe a novel anti-mouse CX3CR1 antibody, clone SA011F11, which is suitable for flow cytometry applications. In addition, to test the biological activity of this antibody in vitro, a chemotaxis assay was performed and the migration of CX3CR1-transfected BA/F3 cells was blocked by this clone with an ED50 of 0.11μg/ml. To test the effect of this antibody in vivo, a group of mice were injected i.p. with SA011F11 for 4 days and the changes in cell populations of different organs (spleen, peritoneal cavity, lungs, brain, and bone marrow) were studied. The administration of clone SA011F11 resulted in the reduction of CX3CR1+ cells in most of the analyzed organs and, although the degree of reduction varied, was more pronounced in the spleen and lungs while showing little effect in the brain. These findings demonstrate that clone SA011F11 has a biological activity in vivo and in vitro, and suggest that it is a powerful tool to study the biology of the CX3CL1/CX3CR1 axis.
Role of LFA-1 for protective immunity elicited by heterologous prime-boost vaccination against intracellular parasitic infection (CCR5P.206)
Authors: Vasconcelos Jose Ronnie , ferreira Camila Pontes , Rodrigues Mauricio
Published: January 2023

Prime-boost vaccine with DNA plasmid followed by adenovirus 5 is a powerful strategy to elicit specific CD8+T cells which play a key role in protective immunity (PI) against intracellular pathogens, such as Trypanosoma cruzi, the causative agent of Chagas' disease. Integrins, CAM and chemokine receptors may also play a role in this process. We demonstrated that treatment with anti-LFA-1 antibody, in immunized/infected mice, reverted the PI after experimental systemic infection. The aim of this work was to study the role of LFA-1 and VLA-4 integrins, the ICAM-1 and CCR5 after prime-boost vaccine. In order to study the role of LFA-1 and VLA-4 integrins, A/Sn mice were immunized with prime-boost vaccine. After, they were infected and treated with anti-LFA-1 and/or anti-VLA blocking antibodies. The immunized/infected mice treated with anti-LFA-1 displayed high parasitemia and all did not survive more than 30 days after challenge. Interestingly, treatment with anti-VLA-4 did not alter the protective response and all mice remained alive. In addition, LFA-1 blockage neither diminished the frequency nor compromised capacity of specific CD8+T cells in producing IFN-γ and/or TNF-α. Also, deficient ICAM-1 or CCR5 mice immunized and/or infected did not become susceptible to challenge. We concluded that LFA-1 play a critical role in the protective immune response generated by the vaccination, and by blocking this molecule, the CD8+ T cell migration capacity is likely impaired.
Soluble IL-15 complexes are generated after CD40 stimulation and VSV infection independent of type I IFN signaling. (CCR6P.218)
Authors: Anthony Scott , Schluns Kimberly
Published: January 2023

Interleukin (IL)-15 is a cytokine that promotes the development and homeostasis of a group of lymphocytes but is also significantly up-regulated in response to immune stimulation and during inflammation. Previous studies show that TLR stimulation and total-body irradiation (TBI) induce soluble IL-15Rα/IL-15 complexes (sIL-15), which are capable of mediating agonistic IL-15 effects in a paracrine manner. The effects of elevated IL-15 are well characterized, yet little is known about the signals inducing these complexes. We report that treatment with αCD40 Ab but not α41BB Ab led to systemic increases in sIL-15 complexes in vivo. BATF3 KO mice, which are deficient in CD8+ DCs, were impaired in the generation of sIL-15 complexes in response to αCD40 treatment, indicating an important role for CD8+ DCs in the generation of sIL-15 complexes in vivo. Interestingly, although CD40 stimulation or VSV infection enhanced systemic IFN-α protein expression, Type I IFN signaling was not required for the induction of sIL-15 complexes during either event. This is in contrast to the requirement for IFNAR signaling in generating sIL-15 complexes after TBI or Poly I:C. CD40 signaling during VSV infection was also not required for sIL-15 complex generation, as blockade with αCD40L exhibited no effect. Overall, this study indicates that sIL-15 complexes are induced in vivo by both type I IFN dependent and independent pathways.
Strong TCR signaling prolongs lineage uncertainty during MHC-I specific positive selection (HEM8P.241)
Authors: Kimura Motoko , Nakayama Toshinori , Singer Alfred
Published: January 2023

CD4/CD8 lineage choice in the thymus is primarily determined by the duration of TCR signaling during positive selection. Positive selection signaling by MHC-I ligands is of relatively short duration because CD8 coreceptor expression is terminated. However, the duration of TCR signaling during MHC-I specific positive selection should, in theory, also be affected by TCR affinity as well as coreceptor signal strength, but how TCR affinity and coreceptor signal strength affect MHC-I specific positive selection is not really known. To address this point, we used RAG-deficient mice expressing a panel of MHC-I restricted TCR transgenes with TCRs of different affinities for in vivo self ligands, and used genetically engineered mouse models that modulate the intensity of TCR signaling. Our data reveal that TCR signal intensity dictates how long MHC-I signaled thymocytes remain lineage-uncommitted before they express Runx3 and undergo CD8 lineage commitment. Furthermore, our data reveal that lineage errors by very high affinity TCR undergoing MHC-I specific positive selection do not normally occur because CD8 coreceptors have evolved to provide only weak Lck/coreceptor signals. We conclude that TCR signal intensity dramatically affects the timing of lineage fate during MHC-I specific positive selection and that error-free CD8 lineage choice is dependent on weak signaling by the CD8 coreceptor.
The hyperinflammatory reactivity of post-asthma alveolar macrophages contributes to RSV-induced asthma exacerbations (HYP7P.269)
Authors: Grooten Johan , Schepens Bert , Smet Muriel , Saelens Xavier , Naessens Thomas
Published: January 2023

Respiratory syncytial virus (RSV) causes serious morbidity as a major etiological agent of allergic asthma exacerbations. We now report that mice that suffered a preceding allergic bronchial inflammation were more prone to develop severe inflammatory responses and AHR after RSV-infection. Post-asthma alveolar macrophages (AM), residing in the alveolar lumen after clearance of eosinophilia, were found to largely contribute to this RSV-induced inflammation exacerbation. This AM population exhibited increased proinflammatory cytokine secretion in response to RSV-challenge but failed to contribute to viral clearance. A selectively increased proinflammatory NF-κB p65 signaling along with unaltered antiviral type I IFN signaling and loss of viral phagocytic clearance appear to be at the basis of the post-asthma AM mediated inflammation exacerbation. Phenotypic analysis revealed that the majority of post-asthma AM consisted of immature SiglecFlowCD11blow/int cells. Full maturation of the immature SiglecFlowCD11blow/int post-asthma AM by intratracheal administration of GM-CSF was associated with a decrease in RSV-induced exacerbation parameters, including pulmonary inflammation and AHR. These results point to a cascade of events in which a prior allergic bronchial inflammation causes a maturation defect in post-asthma AM resulting in a hyperinflammatory reactivity of the cells to RSV-infection, which in turn contributes to the onset of RSV-induced asthma exacerbation.
Development of a novel transcutaneous delivery peptide and its application in skin inflammation (HYP7P.273)
Authors: Kim WonJu , Choi Je-Min
Published: January 2023

Transcutaneous delivery of therapeutic drugs has many advantages over the systemic administration for inflammatory skin diseases such as atopic dermatitis or psoriasis. However, the greatest challenge for transcutaneous delivery is methodology due to the tight junction of skin tissue. Here, we screened and identified transdermal delivery peptide (TDP) from human proteins which would be non-invasive and simple method to deliver various cargos such as chemicals, peptides, proteins, etc. TDP-EGFP recombinant protein was purified and exhibited significantly higher intracellular transduction efficacy compared with TAT-EGFP in Jurkat, HaCaT, NIH3T3 cell lines and also in primary cells including splenocytes and keratinocytes. TDP-TAMRA peptides were successfully delivered into the epidermis and dermis of the mouse ear and back skin. We also used TDP-VIVIT peptide to inhibit T cell activation and contact dermatitis in vivo. TDP-VIVIT significantly inhibited IL-2 secretion with reduction of CD69 and CD44 expression level of activated CD4 and CD8 T cells. Our results suggest TDP-VIVIT peptide could be a potential transdermal applicable therapeutic drug for inflammatory skin diseases.
Overexpression of IL-18 promotes iNKT cell mediated eosinophilic esophageal inflammation in experimental murine model of eosinophilic esophagitis (HYP7P.257)
Authors: Upparahalli Venkateshaiah Sathisha , Jalahalli Mariswamy Siddesha , Shukla Anshi , Mishra Anil
Published: January 2023

Elevated level of IL-18 is reported in a number of allergic diseases and we earlier reported induced blood IL-18 and esophageal IL-18Rα mRNA in human EoE. Therefore, the current study focuses on the hypothesis that allergen-induced IL-18 and its receptor (IL-18Rα) positive cells may have an important role in EoE pathogenesis. Accordingly, we examined the blood IL-18 and esophageal IL-18Rα mRNA levels in aeroallergen and food allergen induced experimental mouse model of EoE. Herein, we demonstrate that blood IL-18 protein and esophageal IL-18Rα mRNA are induced in the mouse model of EoE and the source of induced esophageal IL-18Rα transcript are iNKT cells. We report that intranasal delivery of rIL-18 induces both mast cells and eosinophilic inflammation in the esophagus. Furthermore, to establish the significance of IL-18 in EoE pathogenesis, we next examined DOX-inducible rtTA-CC10-IL-18 bitransgenic mice that have significantly induced IL-18 protein in the esophagus. Our analysis indicated that DOX exposed IL-18 tg mice develop most of the characteristics of EoE, such as esophageal intraepithelial eosinophilia, increased mast cells and esophageal remodeling including fibrosis. Mechanistically, we show that IL-5 gene-deficient mice and iNKT cell-deficient (CD1d null) mice do not induce EoE in response to IL-18 intranasal challenge. Taken together, we provide evidences that allergen induced IL-18 has a significant role in promoting IL-5 and iNKT dependent EoE pathogenesis.
Sphingosine 1-phosphate receptor 3 is highly upregulated on human neutrophils upon migration to the airways (INC6P.315)
Authors: Ingersoll Sarah , Forrest Osric , Brown Milton , Tirouvanziam Rabindra
Published: January 2023

Background and hypothesis: Migration of polymorphonuclear neutrophils (PMNs) from blood into the airway lumen is accompanied by changes in surface receptors and responsiveness to environmental cues, such as sphingolipids. In particular, signaling via extracellular ceramide / sphingosine 1-phosphate (S1P) was suggested to contribute to human airway disease, such as cystic fibrosis (CF). We hypothesized that airway PMNs would alter expression of the S1P receptor, S1PR3, which was recently implicated in lung inflammation and fibrosis. Methods: We collected blood and sputum from healthy control (HC) and CF subjects and analyzed S1PR3 expression in live PMNs gated by flow cytometry. In addition, we used a novel in vitro model to study the effect of transepithelial migration of blood PMNs on S1PR3 expression. Results: We observed a 10-fold upregulation of surface S1PR3 expression on HC and CF airway PMNs, in vivo, compared to their blood counterparts. This major increase was recapitulated in vitro, when naive blood PMNs were induced to transmigrate through an airway epithelial layer toward apical CF airway fluid. S1PR3 expression increased over time, by 10-fold after 10 hours, and up to 20-fold at 18 hours. Conclusions: Modulation of S1PR3 expression follows PMN migration to both HC and CF airways. Further studies will determine the importance of this receptor in mediating responses to ceramide/S1P signaling in these cells, including PMN migration, apoptosis and activation.
Aging-associated chronic mast cell activation by aged mesenteric lymphatic vessels (INC6P.324)
Authors: Gashev Anatoliy , Nizamutdinova Irina , Chatterjee Victor , Cromer Walter , Tobin Richard , Zawieja David , Newell Rogers Karen
Published: January 2023

Here we present novel data related to interactions between lymphatic vessels, mast cells (MCs), eosinophils, and immune cells. MCs are known to inhabit in greater density the tissue near mesenteric lymphatic vessels (MLVs). Acute activation induces MC degranulation with subsequent recruitment of eosinophils and of MHC class II+ cells towards MLVs. Aging permanently influences mesenteric MCs by inducing chronic basal activation, thereby influencing contractility of aged MLVs. At the same time, the number of intact MCs available to react acutely to inflammatory stimuli decreases with age. The diminished number and activity of MCs in aged tissues also diminishes traffic and activation of eosinophils that are attracted to aged MLVs. Concurrently, MCs can function as helper cells for B cells that can produce IgE. IgE has a relatively short half-life in the absence of chronic MC activation. We propose that a key component of aging-associated alterations of “mesenteric lymphatic system-mast cells/eosinophils-inflammatory cells” system is the increased basal activation of resident MCs in aged mesentery that promote chronic inflammation with lesser potential to recruit enough anti-inflammatory MHC class II+ cells and eosinophils towards MLVs, resulting in the inability to prevent a smooth transition from acute inflammation to specific immune cell activation.
Development of uptake capacity and bactericidal activity in myeloid cells during GM-CSF-driven differentiation. (INC6P.326)
Authors: Olatunde Adesola , Landuyt Ashley , Abell Laura , Schwartz Elizabeth
Published: January 2023

The antigen presenting function of dendritic cells (DC) is well defined, yet their ability to handle bacterial infection is poorly understood. We have previously shown that GM-CSF-derived DC can confine Listeria monocytogenes (Lm) to the phagosome, preventing its invasion of the cytosol, growth, and spread. However, it is not clear at what stage of development DC acquire the capacity to retain Lm in the phagosome. To measure this activity at different stages of development, GM-CSF driven bone marrow cells were sorted based on expression of Ly6C, CD115, and CD11c. This strategy enables isolation of cells representing five distinct myeloid cell types: CMP, GMP, monocytes, inflammatory dendritic precursors (IDP), and inflammatory DC (IDC). To measure bacterial invasion of the cytosol, we used an Lm strain that expresses GFP upon cytoplasmic entry, in combination with fluorescent bioparticles to measure bulk uptake. The CMP demonstrated minimal uptake capacity and little bacterial cytoplasmic entry. The GMP population showed greatly increased uptake capacity, but also high susceptibility to bacterial cytoplasmic entry. In the monocyte population, the uptake capacity reached its peak, and these cells gained the capacity to confine the bacteria to the phagosome. Finally, the uptake capacity decreased modestly in the IDP and IDC populations, but these cells also blocked cytoplasmic invasion of the bacteria. The mechanistic basis of phagosomal retention was further examined.
Association of tissue transglutaminase and NLRP3 inflammasome in liver inflammation after Schistosoma japonicum infection (INM6P.343)
Authors: Li Zi , Ji Xiaofang
Published: January 2023

Liver diseases caused by Schistosoma japonicum(Sj) infection is one of the most serious pathological changes. Liver inflammation underlies infection-induced fibrosis, cirrhosis and liver cancer but the processes that promote hepatic inflammation by Sj are not well defined. We indicate that interleukin-1β (IL-1β) confers liver inflammation through Sj-induced NLRP3 inflammasome signaling, and tissue transglutaminase (tTG) is the upstream molecule of this signaling. Five weeks after Sj infection, elevated IL-1β levels in mice liver and also in serum compared to non-infection controls coincide with the expression level and activation of caspase-1 and tTG in mice liver. Immunohistochemical analysis of normal control and Sj-infected mice liver sections revealed that infiltrating inflammatory cells around the granuloma and Hepatic stellate cells (HSCs) among hepatic cells, are the primary cellular source of hepatic IL-1β and caspase-1 p10 during Sj infection. Sj infection triggers MyD88-mediated TLR4 signaling to induce IL-1β mRNA expression. Administration of cystamine, an inhibitor of tTG, abrogates the increase of IL-1β and caspase-1 p10 in infected mice. This result establishes a novel link between tTG and NLRP3 inflammasome under Sj infection. The important function of NLRP3 inflammasome activation and tTG in Sj infection makes them potential drug targets in preventing Sj-induced liver diseases.
Cigarette smoke extracts inhibit TLR ligand-induced inflammation via inactivation of NF-κB and activation of histone deacetylase in macrophages (INM7P.353)
Authors: Lee Kyoung-Hee , Jang An-Hee , Lee Chang-Hoon , Yoo Chul-Gyu
Published: January 2023

Cigarette smoke (CS) is the major risk factor of chronic obstructive pulmonary disease (COPD). CS has been reported to inhibit Toll-like receptor (TLR) function and phagocytosis in macrophages. Moreover, alveolar macrophages in COPD have impaired responsiveness to TLR stimulation. However, the molecular mechanism of CS-mediated impairments of macrophage immunity is not fully defined. We investigated the inhibitory mechanism of cigarette smoke extracts (CSE) in macrophages. Lipopolysaccharide (LPS, TLR4 ligand), Pam3CSK4 (TLR2 ligand), and CpG-oligodeoxynucleotides (TLR9 ligand) induced IL-6, TNF-α, and IL-1β production in RAW264.7 macrophage cells, which was significantly suppressed by CSE co-treatment. CSE did not block LPS-induced IκBα degradation, MAP kinase activation, and nuclear translocation of NF-κB subunit. However, it markedly suppressed mRNA expression of IL-6, TNF-α, and IL-1β, which implies that CSE regulate inflammatory cytokine production at the transcriptional level. Therefore, we next investigated the activity and modification of transcription factor, NF-κB, and histone deacetylase (HDAC). CSE significantly suppressed basal and LPS-induced NF-κB activity and reduced the level of acetyl-NF-κB p65. CSE increased HDAC activity and decreased the level of acetyl- and phospho-Histone H3 (Lys9/Ser10) expression. Together, these results suggest that CSE-mediated impairments of macrophage immunity are mediated by inhibition of NF-κB activation and activation of HDAC.
CD13 restricts TLR4 endocytic signal transduction in inflammation (INM7P.350)
Authors: Ghosh Mallika , Subramani Jaganathan , Rahman Mamunur , Shapiro Linda
Published: January 2023

The Toll-like Receptor 4 (TLR4) is the prototypical sensor of infection or injury that orchestrates the innate response via sequential activation of both cell-surface and endocytic signaling pathways that trigger distinct downstream consequences. Positive and negative regulators of this process exist to maintain the critical balance required for resolving inflammation. Myeloid CD13 negatively modulates receptor-mediated antigen uptake to control T cell activation in the adaptive immune response. Here we report that CD13 governs the internalization of TLR4 and subsequent innate signal transduction cascades leading to IRF3 activation independently of CD14, a positive regulator of TLR4. In response to LPS, CD13 is co-internalized with TLR4, CD14 and dynamin into Rab5+ early endosomes. Importantly, pIRF-3 activation and transcription of its targets are enhanced in CD13KO cells, while TLR4 surface signaling remains unaffected resulting in a skewed inflammatory response. Further, ischemic injury in vivo provoked identical TLR4 responses. Finally, CD13KO mice showed significantly enhanced type I Interferon-mediated signal transduction, escalating inducible nitric oxide synthase transcription levels and promoting accumulation of oxidative stress mediators and tissue injury. In response to injury or infection, CD13 negatively regulates TLR4 signal transduction, thereby balancing the innate response by maintaining the inflammatory equilibrium critical to innate immune regulation.
B4GALT6 regulates astrocyte activation during CNS inflammation (INM8P.360)
Authors: Mayo Lior , Trauger Sunia , Blain Manon , Mascanfroni Ivan , Kivisäkk Pia , Yeste Ada , Bakshi Rohit , Alvarez Jorge Iván , Prat Alexandre , Antel Jack , Weiner Howard , Quintana Francisco
Published: January 2023

Astrocytes play complex roles in neuroinflammation. Thus, it is important to characterize the mechanisms regulating astrocyte function, as well as potential targets for the therapeutic modulation of astrocyte activity. Here we report that during the chronic-progressive phase of EAE, astrocytes promote disease pathogenesis by a lipid-dependent signaling pathway. In line with previous studies, depletion of reactive astrocytes during the acute phase of chronic-progressive EAE exacerbated disease. However, depletion of astrocytes in the chronic phase ameliorated the disease. Hence, to understand this dichotomy we compared gene expression profiles of astrocytes from mice in acute and chronic stages of EAE. One gene associated with the chronic stage was B4GALT6, which codes for the β-1,4-galactosyltransferase 6 that catalyzes the synthesis of lactosylceramide (LacCer). We observed that LacCer levels are upregulated in the CNS during chronic EAE, and that LacCer synthesized by the astrocytes acts in an autocrine manner to trigger transcriptional programs that promote the recruitment and activation of infiltrating monocytes and microglia, and neurodegeneration. We also detected increased B4GALT6 expression and LacCer levels in MS lesions. Finally, the inhibition of LacCer synthesis suppressed CNS innate immunity and neurodegeneration, and interfered with the activation of human astrocytes. Thus, B4GALT6 is a potential therapeutic target for MS and neuroinflammatory disorders.
The extracellular adherence protein from Staphylococcus aureus inhibits the classical and lectin pathways of complement by blocking formation of the C3 proconvertase (INM8P.361)
Authors: Woehl Jordan , Stapels Daphne , Garcia Brandon , Ramyar Kasra , Keightley Andrew , Ruyken Maartje , Sfyroera Georgia , Weber Alexander , Zolkiewski Michal , Ricklin Daniel , Lambris John , Rooijakkers Suzan , Geisbrecht Brian
Published: January 2023

The pathogenic bacterium Staphylococcus aureus actively evades many aspects of human innate immunity by expressing a series of small inhibitory proteins. A number of these proteins inhibit the complement system, with the majority of S. aureus acting on the alternative pathway, blocking the amplification loop. Only a few proteins act on the initial recognition cascades that constitute the classical pathway (CP) and lectin pathway (LP). By screening a collection of recombinant, secreted staphylococcal proteins, we identified the extracellular adherence protein (Eap) as a potent, specific inhibitor of both the CP and LP. We found that Eap blocked CP/LP-dependent activation of C3, but not C4, and that Eap likewise inhibited deposition of C3b on the surface of S. aureus cells. In turn, this significantly diminished the extent of S. aureus opsonophagocytosis and killing by neutrophils. This combination of functional properties suggested that Eap acts specifically at the level of the CP/LP C3 convertase (C4b2a). Indeed, we demonstrated a direct, nanomolar-affinity interaction of Eap with C4b. Eap binding to C4b inhibited binding of both full-length C2 and its C2b fragment, which indicated that Eap disrupts formation of the CP/LP C3 proconvertase (C4b2). As a whole, our results demonstrate that S. aureus inhibits two initiation routes of complement by expression of the Eap protein, and thereby define a novel mechanism of immune evasion.
Intranasal or intraperitoneal administration of human tubal mesenchymal stem cells reduces T lymphocytes-associated lung inflammation in cigarette-induced emphysema in mice (IRC10P.415)
Authors: Peron Jean Pierre , de Brito Auriléia , Rigonato Nicole , Vitoretti Luana , Moraes Gabriel , Soares Clariana , Czeresnia Carlos Eduardo , Perin Paulo , Maluf Mariângela , Zatz Mayana , Ligeiro de Oliveira Ana Paula
Published: January 2023

Objective: To study the effectiveness of human tubal Estromal Stem Cells (htESCs) therapy by intra-nasal or intra-peritoneal route in experimental cigarette smoke-induced emphysema in C57BL/6 mice. Design: To achieve that, C57BL/6 mice were exposed to cigarette smoke for 75 days (2 times / day) and received or not two doses of htMSCS either intraperitoneally or intranasally. Experiments were performed on day 76th. Materials and Methods: Briefly, brochoalveolar lavage fluid was obtained and either used to measure cytokine secretion or inflammatory infiltrate. Moreover, lung tissue was also submitted to imunohistochemistry for the transcription factors NF-AT and NF-kB and also IL-10. Results: Our results clearly show that htMSCs greatly reduced lung inflammation, with decreased cellular infiltrate of T CD4 and T CD8 lymphocytes, which was confirmed by reduced NF-AT staining in lung tissues. This was associated with reduced pro-inflammatory cytokine secretion, as TNF-a, IL-1b and IL-6, less mucus and collagen deposition, and interestingly, increased IL-10. Conclusion: Altogether, our data indicate that therapy with htMSCs for the treatment of emphysematous disease may be a promising clinical approach. Moreover, intranasal route is also effective, and must taken in consideration due to its lack of invasiveness.
NKT cell-mediated enhancement of humoral immunity against Clostridium difficile toxin B (IRC10P.406)
Authors: Rampuria Pragya , Devera Thomas , Lang Mark
Published: January 2023

To develop more effective antibody-stimulating vaccines, we must improve our understanding of the mechanisms by which vaccines induce and maintain long-lasting humoral immunity. This is important for the development of new vaccines against emerging pathogens such as C. difficile. It is well-established that Natural Killer T (NKT) cells, activated with the glycolipid, α-galactosylceramide (α-GC) enhance humoral immunity, but the mechanisms by which they do so are poorly understood. Here we show that activated NKT cells enhance the antigen-specific IgG1 response and survival against C. difficile Toxin B (TcdB). We and others have shown that NKT cells may enhance priming of T helper (Th) cells. We therefore hypothesized that T helper and NKT subsets coordinate to enhance the humoral immune response against C. difficile. Flow cytometry analysis demonstrated that NKT cell activation with a-GC did not alter numbers of Th or follicular T helper (Tfh) cells, but increased the number of NKTfh cells. ELISPOT assays indicated that the α-GC enhanced secretion of IL-4 and IL-21 is mainly Th-derived. Adoptive transfer experiments show that α-GC did not alter the ability of Th cells to boost antibody production. This suggests that NKT cells may exert direct and indirect effects (via Th cells) on humoral immunity. Understanding the mechanisms by which NKT cells enhance humoral immunity will help in designing strategies to appropriately activate NKT cells and boost vaccine efficacy.
CTLA-4-mediated suppressor effect by human T follicular regulatory cells (IRC11P.432)
Authors: Sayin Ismail , Herati Ramin , Wherry E. , Betts Michael , Canaday David
Published: January 2023

T follicular helper (Tfh) cells, a subset of CD4 T cells, help B cells produce high affinity class switched antibodies and are essential for generation of germinal centers (GC). T follicular regulatory cells (Tfr) are a newly defined regulatory CD4 T cell subset that suppresses GC formation and B cell function through CTLA-4 dependent mechanisms in murine models. We will test the hypothesis in human systems that Tfr have significant regulatory effects on antibody production in a CTLA-4 dependent manner. Tfh and Tfr cells from human mesenteric lymph nodes were analyzed using flow cytometry. Tfr were defined as CXCR5+, PD-1bright, FoxP3+ cells that were also found to be Bcl6+, CD25+, CD127dim, ICOS+. Flow sorted Tfr and Tfh cells were used to determine the effect of Tfr on Tfh-mediated B cell production of IgG. Tfh cells with mitogen helped B cells produce high amounts of IgG whereas addition of Tfr to this co-culture decreased the IgG levels >90%. Tfh/Tfh ratio directly impacts levels of suppression. The mechanism of the suppressive effect of Tfr cells was investigated by neutralizing IL-10, TGF-β RII and CTLA-4 in the co-culture functional assay. Only anti-CTLA-4 affected IgG production. The present study in the human system suggests that Tfr cells suppressed Tfh-induced antibody production by B cells with CTLA-4 playing an important role in this suppression. These regulatory interactions may play a role in autoimmune diseases and humoral response to vaccines and pathogens.
AhR acts as a Foxp3-independent transcription factor for the suppression of type 1 diabetes in NOD mice (IRC11P.425)
Authors: Ehrlich Allison , Pennington Jamie , Wang Xisheng , Kerkvliet Nancy
Published: January 2023

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor associated with immunosuppression. We previously reported that suppression of type 1 diabetes by TCDD, a potent AhR ligand, is associated with an increased percentage of Foxp3+ regulatory T cells. Similar associations between AhR activation and increased Foxp3+ cells have been reported for suppression of other autoimmune diseases; thus, AhR is a potential therapeutic target. We recently discovered a novel, rapidly metabolized AhR ligand, 10-chloro-7H-benzimidazo[2,1-a]benzo[de]iso-quinolin-7-one (10-Cl-BBQ), that activates AhR in CD4+ T cells, in a manner similar to TCDD. Here we show oral treatment with 10-Cl-BBQ is highly effective in suppressing pancreatic islet infiltration through 20 weeks of age in NOD mice. When Foxp3+ cells were assessed, a small yet significant increase in the frequency, but not total number, of cells was observed. This raised the yet unanswered question, is the increase in Foxp3+ Tregs driving AhR-mediated immune suppression? To directly assess the requirement of Foxp3+ cells in AhR induced suppression of islet infiltration, transgenic NOD.Foxp3DTR mice were employed. In control mice, Foxp3 depletion led to rapid pancreatic islet infiltration. However, treatment with 10-Cl-BBQ suppressed infiltration even in the absence of Foxp3+ cells. This suggests that AhR functions as a Foxp3-independent transcription factor driving suppression of T cell dependent immune responses.