bluuebunny/crossref_metadata_2025 huggingface dataset:
FOXP3 isoform expression in atherosclerosis: correlations with plaque inflammation and disease outcome. (P4140)
Authors: Joly Anne-Laure , Liu Sang , Mailer Reiner , Andersson John
Published: January 2023

Atherosclerosis, a chronic inflammatory disease of arteries, is the leading cause of cardiovascular diseases. Innate as well as adaptive immune responses contribute to atherogenesis, with components of cholesterol carrying low-density lipoprotein triggering inflammation, T-cell activation and antibody production during the course of disease. Regulatory T cells (TRegs), known to suppress immune responses and regulate peripheral tolerance, have been shown to attenuate atherosclerosis in experimental models. The transcription factor Forkhead box P3 (FOXP3) protein is the lineage specification factor of TRegs and plays a critical role in upholding the suppressive activity of TRegs. Three splice forms of FOXP3 mRNA exist in human given birth to isoforms with different functional properties. Full length FOXP3 and FOXP3Δ2 confer a suppressive phenotype to TReg cells whereas FOXP3Δ2Δ7 inhibits the function of the other FOXP3 isoforms in a dominant negative manner. Herein we define the relation between FOXP3 isoform expression and the inflammatory response as characterized by cell markers, cytokine expression and expression of costimulatory molecules in human atherosclerotic plaques. We also correlate FOXP3 isoform expression with cardiovascular events post-endarterectomy.
Characterization of the function and co-expression patterns of human FOXP3 isoforms and their role in IPEX syndrome development. (P4146)
Authors: Darrasse-Jeze Guillaume
Published: January 2023

IPEX syndrome is a rare and fatal X-linked recessive disorder due to mutations in the FOXP3 gene, which plays a critical role in the development and function of the regulatory T cells (Tregs) that maintain self-tolerance. Contrary to mice, humans possess three isoforms of FOXP3 with unknown function, and the expression of these isoforms is not restricted to Tregs. We determined the levels of expression and distribution of FOXP3 and other transcription factors isoforms in normal human T-cell subsets and in T cell subsets of patients with autoimmunity at the single-cell level by multiplex RT-qPCR and Flow cytometry and observed that expression of these isoforms is not restricted to Tregs, but can also be detected in naïve and activated/memory helper (Th) and in activated/memory CD8+ T cells. Distinct patterns of co-expression of the FOXP3 isoforms are modulated in response to inflammatory or autoimmune stimuli. Moreover, we observed that normal FOXP3 isoforms could induce bona fide regulatory function in Th cells. Our work supports the view that lack of FOXP3 expression in the effector T-cell populations in IPEX patients may contribute to the pathogenesis of the disease and suggests that FOXP3 deficiency can be corrected by gene transfer of the appropriate FOXP3 isoforms into conventional T-cells, which has important implication for the treatment of IPEX patients.
Realtime immune cell interactions in target tissue during immune damage and protection (P4112)
Authors: Miska Jason , Abdulreda Midhat , Devarajan Priyadharshini , Lui Jen Bon , Suzuki Jun , Pileggi Antonello , Berggren Per-Olof , Chen Zhibin
Published: January 2023

Realtime imaging studies are reshaping immunological paradigms, but a visual framework is lacking for the cellular dynamics of self-antigen-specific T cells at the effector phase in target tissues. To delineate key events in the cascade of tissue damage, immune protection and tissue response, we conducted noninvasive longitudinal imaging at a cellular resolution. We report that for both CD4+ and CD8+ effector T (Teff) cells, destruction was associated with close proximity to targets. Antigen-specificity is a prerequisite of target killing. Strikingly, incongruent to a classic concept of bystander killing, juxtaposed β-cell mass lacking the specific antigen was not subject to destruction, but grew in days, likely by replication. In the target tissue, Foxp3+ regulatory T (Treg) cells persistently contacted Teff cells, providing the first in vivo evidence consistent with the in vitro “trademark” activity of Treg cells, contact-dependent suppression. Modulating cytotoxic T lymphocyte antigen-4 (CTLA4) altered Treg-Teff cell interactions and unleashed Teff cells to inflict further damage. This study demonstrates the importance of contact-based immune cell interaction and suggests that an intimate choreography between destructive and protective immune cells is critical for tolerance induction in target tissues. It also highlights the potential of tissue regeneration under antigenic incognito in inflammatory settings.
Hijacking of microparticle transcellular communication: a means of protein delivery and alteration of cell function (P3257)
Authors: Sahler Julie , Woeller Collynn , Spinelli Sherry , Blumberg Neil , Phipps Richard
Published: January 2023

Microparticles are submicron vesicles present in blood and other fluids that influence cells such as lymphocytes, monocytes, endothelial cells, etc. Microparticle composition is derived from the parent cell and determines the “message” communicated to other cells. Platelets and megakaryocytes produce about 80% of circulating microparticles. We aimed to usurp this unique means of transcellular communication by manipulating microparticle composition in order to deliver proteins to target cells to influence their function. We used a lentiviral vector for the transduction of a human megakaryoblastic cell line, thus changing the protein composition of generated platelets and microparticles. We successfully engineered microparticles to overexpress the anti-inflammatory transcription factor peroxisome proliferator activated receptor-γ (PPARγ) along with GFP. These genetically enhanced microparticles were taken up by the monocytic cell line THP-1 and by primary endothelial cells, and this uptake was enhanced by PMA activation. Importantly, the transfer of PPARγ was functional, in that, microparticles that overexpressed PPARγ caused THP-1 cells to increase transcription of a PPARγ target gene, fatty acid binding protein 4. This proof of concept alteration of microparticle composition and subsequent functional changes in target cells provide an important new tool for therapeutic delivery of proteins, which could be developed into new anti-inflammatory or adjuvant therapies.
Segmentation: a novel approach to immune profile analysis of the human lymph node microenvironment (P3261)
Authors: Blenman Kim , Lee Peter
Published: January 2023

In the lymph node microenvironment, many cells interact to provide cues for immune cell activity. Understanding the composition and spatial relationships between cells is key in deciphering mechanisms responsible for immune responses. Our novel method expands the use of immunohistochemistry (IHC) by generating quantitative immune profiles. Our integrated approach includes multiplexed IHC, high-resolution multispectral automated whole-slide scanning, and image analysis via software. Our image analysis segments cells into phenotypes and tissues into regions. In this pilot study, we compared tumor-infiltrated (Tpos) and tumor-free (Tneg) tumor-draining lymph nodes (TDLNs) from breast cancer patients. We segmented images into non-immune cell (NIC), extrafollicular (ExF), follicular (F), and tumor tissue regions. We segmented cells by five-color IHC into T cell (CD4, CD8, and Foxp3) and tumor (cytokeratin) phenotypes. Globally and compartmentally (NIC, ExF, and Tumor), there were more immune cells in Tpos TDLNs. However, in F regions, there were less immune cells in Tpos TDLNs. There were more NIC regions but less ExF and F regions in Tpos TDLNs. We also found less NIC, ExF, and F regions in relapsed vs. disease-free patients. Our segmentation analysis may provide valuable data on cell distribution and spatial relationships within specific tissue regions. Our next steps are to use this method to help define mechanisms of action for tumor metastasis and predict clinical outcome.
Automatic pathogen introduction and removal for live cell imaging of host-pathogen interaction (P3237)
Authors: Hung Paul
Published: January 2023

Host-pathogen interactions represent a significant area of immunology, encompassing the study of how organisms cause infections and diseases. As the field advances, there is increasing interest in using in vitro cell culture models and imaging to study the interactions. However, a few barriers remain with the conventional static cell culture approach. Not only the pathogen will easily outgrow the cells over time, prohibiting further studies, but tedious liquid exchange steps are required to remove excessive pathogens or to add other reagents such as therapeutic compound. To facilitate host-pathogen interaction research with live cell imaging, a microfluidic platform which addresses the problems mentioned above was developed. The platform allows the researchers to culture the cells in a microchamber on a standard inverted microscope, as well as preload up to four different reagents which can be introduced to the cells automatically including the pathogen. The reagent switching function enable the removal of pathogens from the chamber after incision, and subsequent monitoring of host cell response over up to 3 days. The enclosed small volume microchamber also provides practical advantages for working with infectious agents during live cell imaging. An experiment using human intestinal cells infected with engineered E. coli strains was demonstrated. Both an invasive and a non-invasive bacterial strain were monitored for infection with time-lapsed imaging up to 24 hours.
R10K update: NGS of immune repertoire for biomarker discoveries (P3241)
Authors: Han Jian , Wang Chunlin , Byrne-Steele Miranda , Alleyne Jessica , Yang Qunying , Myers Richard
Published: January 2023

The natural duty of the adaptive immune system is to detect any internal and external threats and mount specific and measured reactions accordingly. Therefore, the immune repertoire is rich in specific diagnostic markers. We have developed a novel multiplex PCR method, which can amplify the CDR3 regions from a sample inclusively and semi-quantitatively. The PCR products can be sequenced directly with different next generation sequencing platforms. A software package was developed to filter out amplification and sequencing errors, identify CDR3 sequences and perform routine repertoire analysis. The R10K project (www.R10K.org) is organized and supported by the HudsonAlpha Institute for Biotechnology, a nonprofit research institute specialized in genomic research. The project is an international collaborative effort to sequence 10,000 samples and study 100 diseases. Our pilot studies have demonstrated that disease specific repertoire changes can be detected. Quantitatively, the changes in repertoire diversity can be used as a biomarker for disease staging and measuring treatment response. Qualitatively, detection of disease specific CDR3s can be used for early diagnosis of diseases, including cancer, autoimmune diseases, inflammatory diseases, and infectious diseases. The R10K project is open to collaborators; the IP rights are shared among collaborators, participating institutions and companies. The data will be publicly available as each study is completed.
Posttranscriptional gene regulation of IL-17 by the RNA-binding protein HuR is required for initiation of autoimmune neuroinflammation (P6273)
Authors: Chen Jing , Cascio Jason , Magee Joseph , Techasintana Patsharaporn , Gubin Matthew , Garrett Dahm , Calaluce Robert , Atasoy Ulus
Published: January 2023

Interleukin 17 (IL-17) is a proinflammatory cytokine produced by activated Th17 T cells. Th17 cells are major contributors to autoimmune diseases, such as multiple sclerosis. Although the transcriptional regulation of Th17 cells is well understood, its posttranscriptional regulation is unclear. The RNA-binding protein HuR regulates the stability of many target mRNAs via binding the AU-rich elements (ARE) present in the 3’ untranslated region (UTR), including inflammatory cytokines such as IL-4, IL-13 and TNF-α. The regulation of IL-17 expression by HuR has not been established. We reported that production of mRNA and protein levels of IL-17, produced by polarized Th17 cells from HuR conditional knockout (KO) mice, were abrogated. Moreover, we demonstrated that HuR directly bound to IL-17 mRNA 3’ UTR by using immunoprecipitation and RNA precipitation (RIP) and biotin pull down assays. Th17 cells from HuR conditional KO mice had decreased IL-17 steady-state mRNA and protein levels compared to wild type cells. Mice with adoptively transferred HuR KO CD4+ T cells had delayed onset and reduced severity of experimental autoimmune encephalomyelitis (EAE) compared to wild type CD4+ T cells. Our results reveal a posttranscriptional regulatory mechanism of Th17 cells by HuR, which may provide novel therapeutic targets for treatment of IL-17- mediated autoimmune neuroinflammation.
IFN-γ signaling in astrocytes and microglia has opposite roles in CNS autoimmunity (P5170)
Authors: Zhang Guang-Xian , Yan Yaping , Ding Xiaoli , Li Ke , Ciric Bogoljub , Yang Jingxian , Wu Shuai , Xu Hui , Chen Wanjun , Lovett-Racke Amy , Rostami Abdolmohamad
Published: January 2023

IFN-γ, the hallmark cytokine of Th1 cells, plays an important role in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Thus far, the role of IFN-γ in EAE has been largely studied through its effects on immune cells, while much less is known about its effects on central nervous system (CNS) cells, especially in vivo. In this study we for the first time dissected the in vivo effects and mechanisms of IFN-γ signaling in astrocytes and microglia and found that IFN-γ signaling in these cell types has opposite effects in EAE pathogenesis. Silencing IFN-γ signaling in astrocytes ameliorated EAE, while in microglia it increased disease severity. Silencing IFN-γ signaling in astrocytes resulted in diminished expression of chemokines and fewer inflammatory cells infiltrating into the CNS, while blocking IFN-γ signaling in microglia increased disease severity through augmented activation and proliferation of microglia. Further, blocking IFN-γ signaling in astrocytes ameliorated both Th1- and Th17-mediated adoptive EAE, indicating an important role for IFN-γ signaling in astrocytes in autoimmune CNS inflammation. Thus, our study defines novel mechanisms of action of IFN-γ in EAE pathogenesis, and also highlights an opportunity for development of MS therapies directed at CNS cells.
Reciprocal modulation of CD4 T cell responses by intravenous immunoglobulin in experimental autoimmune encephalomyelitis (P5178)
Authors: Othy Shivashankar , Hegde Pushpa , Topçu Selma , Sharma Meenu , Maddur Mohan , Lacroix-Desmazes, Sebastien , Käsermann Fabian , Miescher Sylvia , Bayry Jagadeesh , Kaveri Srini
Published: January 2023

IVIg is increasingly used for therapy in many autoimmune and inflammatory diseases; however the mechanisms underlying its therapeutic benefit have remained uncertain. We have investigated the mechanisms in the context of modulation of regulatory and pathogenic CD4 T cells in EAE, a murine model of multiple sclerosis. We demonstrate that IVIg delays the onset of EAE by inhibiting encephalitogenic CD4 T cell subsets (Th1 and Th17 cells) and concomitantly expanding foxp3+ regulatory T cells. Furthermore, IVIg renders effector T cells less pathogenic by decreasing the expression of encephalitogenic molecules such as GM-CSF and podoplanin. Intriguingly and contrary to the current arguments, the inhibitory FcγRIIB and sialylation on IgG are dispensable and F(ab’)2 fragments retained this function of IVIg. Additionally, IVIg sequestered effector T cells into the draining lymph nodes leading to decreased infiltration into CNS. IVIg interfered with S1P-S1P1-mTOR pathway to sequester lymphocytes in the draining lymph nodes and reciprocally regulate CD4 T cell subsets. Our study reveals a novel role of immunoglobulins in modulating polarization, pathogenicity and trafficking of T lymphocytes accounting for the observed beneficial effect during IVIg therapy.
Regulatory B cells suppress experimental autoimmune myasthenia gravis via production of IL-10 (P5145)
Authors: Sheng Jianrong , Prabhakar Bellur , Meriggioli Matthew , Soliven Betty
Published: January 2023

Autoimmune Myasthenia Gravis (MG) is T cell-dependent, B cell mediated autoimmune disorder in which autoantibodies are produced targeting the skeletal muscle acetylcholine receptor (AChR). Previous studies have shown that regulatory T cells (Tregs) have the capacity to prevent and reverse experimental autoimmune MG (EAMG). In more recent investigations, our studies showed that regulatory B cells (B10 cells) produced high level of IL-10 and suppressed ongoing experimental autoimmune MG (EAMG), resulting in a significant down-modulation of relevant pathogenic anti-AChR autoantibody production and significant preservation of muscle AChR content. In in vitro co-culture studies, B10 cells altered T cell cytokine profiles through increasing IL-10 production and decreasing IFN-r production of CD4+ T cells, but did not directly inhibit T cell proliferation. On the other hand, B10 cells can inhibit B cell proliferation and its autoantibody production in vitro through IL-10 production. Thus, Our studies has provided the significant insights into the nature of the mechanism and effects of regulatory B cells in EAMG. These observations suggest that mobilization of B10 cells in vivo/vitro may represent an effective strategy in the treatment of human MG.
MIF contributes to Type 1 diabetes favors insulitis by mediating macrophages and dendritic cells maturation (P4533)
Authors: Sanchez Zamora Yuriko , Rodriguez Sosa Miriam
Published: January 2023

Type 1 diabetes (T1D) is characterized by a cellular infiltrate in pancreatic islets where β cells are destroyed. The recognition of antigens and auto antigens takes place by macrophages (Mo) and dendritic cells (DCs). Previously we have showed that MIF favors the expression of co-stimulatory molecules on Mo and DCs on infection diseases. However, the role of MIF on Mo and CDs in T1D has not been explored. Here, we determined the expression of co-stimulatory molecules on Mo and CDs from WT or MIF-/- mice on experimental T1D induced by STZ. Cells extracted from pancreas and spleens were treated with antibodies anti- CD80, CD86, CD40, MHC-II and TLRs. Also we tested the number of CD4+CD25+FOXp3+ cells. The results show that MIF-/- mice not developed high glucose levels compared with WT mice. Pro-inflammatory cytokines in serum were diminished on MIF-/-STZ mice, while the anti-inflammatory cytokines were higher compared to WT STZ mice. Moreover, MIF-/-STZ mice had less CD80, CD86, MHCII, TLR-2 and TLR-4 in spleen and pancreatic islets Mo and CDs. In addition, CD4+CD25+ spleen cells had a high expression of FOXp3 transcription factor in MIF-/- healthy than MIF+/+ healthy mice. 8 weeks after of STZ induction the MIF-/-STZ mice present higher FOXp3 expression than WT STZ mice. Our results suggest that MIF favors the expression of co-stimulatory molecules in the Mo and CDs in T1D. Additionally we propose that MIF down-regulate the proliferation of regulatory T cells in T1D.
Determination of anti-PF4/heparin complex antibodies in the fluid phase (P4549)
Authors: Schulze Annika , Alahmad Adnan , Krauel Krystin , Jensch Inga , Greinacher Andreas , Hundt Matthias
Published: January 2023

Antibodies against the human platelet factor 4/heparin complex (PF4/H) are found in the immune-mediated adverse drug reaction heparin-induced thrombocytopenia. These antibodies are directed against PF4 within the complex due to conformational changes of PF4. To study the humoral immune response to PF4/H in greater detail, C57BL/6 mice were immunized with murine (mu) PF4/H by retro-orbital injection. Serum was obtained, and analyzed by ELISA either with muPF4/H directly plate-bound (solid phase), or in a fluid phase assay with muPF4/H that contained 10% biotinylated muPF4. The two approaches differed notably in their specificity and sensitivity to detect anti-muPF4/H antibodies. The solid phase ELISA could not distinguish between antibodies against plate-bound muPF4 alone and plate-bound muPF4/H. Only incubation with excess heparin allowed to distinguish these antibodies. In contrast, the fluid phase ELISA permitted the direct distinction between anti-muPF4 and anti-muPF4/H antibodies. The fluid phase system was also successfully applied to detect muPF4/H-specific B cells by ELISpot. Thus, the fluid phase ELISA for the detection of anti-muPF4/H antibodies was superior to the solid phase assay. Furthermore, our data suggest that surface binding of PF4 might induce conformational changes similar to those induced by heparin binding. Finally, fluid phase ELISAs might be preferable for the detection of antibodies that bind to conformation-dependent epitopes.
Serum amyloid A stimulates macrophage foam cell formation via upregulation of lectin-like oxidized low-density lipoprotein receptor 1 (P5188)
Authors: Jung Young Su , Lee Ha Young , Lee Sung Kyun , Kim Sang Doo , Baek Suk-Hwan , Bae Yoe-Sik
Published: January 2023

Elevated levels of serum amyloid A (SAA) is a risk factor for cardiovascular diseases, however, the role of SAA in the pathophysiology of atherosclerosis remains unclear. Here we show that SAA induced macrophage foam cell formation. SAA-stimulated foam cell formation was mediated by c-jun N-terminal kinase (JNK) signaling. Moreover, both SAA and SAA-conjugated high density lipoprotein stimulated the expression of the important scavenger receptor lectin-like oxidized low-density lipoprotein receptor 1 (LOX1) via nuclear factor-kB (NF-kB). A LOX1 antagonist carrageenan significantly blocked SAA-induced foam cell formation, indicating that SAA promotes foam cell formation via LOX1 expression. Our findings therefore suggest that SAA stimulates foam cell formation via LOX1 induction, and thus likely contributes to atherogenesis.
The tandem PH domain-containing protein 2 regulates chemokine-induced cytoskeletal reorganization and malignant B cell migration (P5194)
Authors: Li Hongzhao , Hou Sen , Wu Xun , Nandagopal Saravanan , Lin Francis , Kung Sam , Marshall Aaron
Published: January 2023

The tandem PH domain-containing protein 2 (TAPP2), along with its homologue TAPP1, is best known for high-specificity binding to PI(3,4)P2, a lipid product of phosphoinositide 3-kinases (PI3K). While PI3K enzymes have a number of functions in cell biology, including cell migration, the functions of PI(3,4)P2 and its binding proteins are not well understood. Previously we found that TAPP2 is highly expressed in leukemic B cells with strong migratory capacity. Here we show that the migration of human malignant B cells is significantly inhibited by TAPP2 knockdown (KD), manifested as reduction in percentage of migrating cells, migration velocity and directionality. TAPP2 KD led to alterations in chemokine-induced rearrangement of the actin cytoskeleton and failure to form polarized morphology. TAPP2 co-localized with the stable F-actin-binding protein utrophin, with both molecules reciprocally localizing against F-actin accumulated at the leading edge. In TAPP2 KD cells, Rac was over-activated and localized to multiple membrane protrusions, suggesting that TAPP2 may act in concert with utrophin and stable F-actin to spatially restrict Rac activation and reduce formation of multiple membrane protrusions. In summary, this study identified TAPP2 as a novel regulator of malignant B cell migration and a potential therapeutic intervention target.
ER stress and neuroinflammation: connecting the unfolded protein response to JAK/STAT signaling (P5196)
Authors: Meares Gordon , Benveniste Etty
Published: January 2023

Neuroinflammation and endoplasmic reticulum (ER) stress are associated with many neurological diseases. ER stress is brought on by misfolded proteins. In turn, cells respond with activation of the unfolded protein response (UPR). The UPR is a highly conserved pathway that transmits both adaptive and apoptotic signals to restore homeostasis or eliminate the irreparably damaged cell. Recent evidence indicates that ER stress and inflammation are linked. In this study, we have examined the interaction between ER stress and JAK/STAT-dependent inflammation in astrocytes. The JAK/STAT pathway mediates the biological actions of many cytokines and growth factors. We have found that ER stress leads to the activation of STAT3 in a JAK1-dependent fashion. ER stress-induced activation of the JAK1/STAT3 axis leads to expression of IL-6 and several chemokines. The activation of STAT3 signaling is dependent on the protein kinase PERK, a central component of the UPR. Knockdown of PERK abrogates ER stress-induced activation of STAT3 and overexpression of PERK is sufficient to activate STAT3. Additionally, ER stressed astrocytes, via paracrine signaling, can stimulate activation of microglia leading to production of oncostatin M (OSM). OSM can then synergize with ER stress in astrocytes to drive inflammation. Together, this work describes a new PERK-JAK1-STAT3 signaling pathway that may elicit a feed-forward inflammatory loop involving astrocytes and microglia to drive neuroinflammation.
Host macrophages are a barrier to the engraftment of embryonic stem cell-derived hematopoietic progenitors. (P4544)
Authors: Thompson Heather , McLelland Bryce , van Rooijen Nico , Manilay Jennifer
Published: January 2023

Embryonic stem cell-derived hematopoietic progenitors (ES-HPs) have been proposed as an alternative bone marrow (BM) source. ES-HPs display features of adult hematopoietic progenitors, but do not achieve high levels of engraftment after transplantation, even in immunocompromised hosts. We hypothesized that host macrophages (Mϕ) are a barrier to ES-HP engraftment. To test this, we cultured and transplanted CD41+ ES-HPs into sublethally irradiated NOD.SCID.γc (NSG) mice that lack B, T and NK cells, but still possess Mϕ. Less than 5% ES-HP donor chimerism in the BM and spleen was observed in NSG recipients, in contrast to >90% chimerism in adult BM-transplanted controls. In addition, we noticed a 3.5-fold increase in spleen weight and a 1.75-fold increase in splenic host F4/80+ Mϕ in ES-HP recipients. In vitro phagocytosis assays confirmed that allogeneic Mϕ phagocytosed ES-HPs at higher levels compared to adult Lin- BM targets. Interestingly, syngeneic Mϕ also engulfed ES-HPs, but not adult Lin- BM. When Mϕ were eliminated with liposome-encapsulated clodronate, a 2.17-fold increase in ES-HP engraftment in the BM and 5.14-fold increase in the spleen was observed. Taken together, our data demonstrate that F4/80+ Mϕ are a barrier to ES-HP engraftment and suggest that cultured ES-HPs are unable to regulate host Mϕ phagocytosis. In line with this, ES-HPs expressed low levels of the Mϕ inhibitory ligand, CD47. Functional studies using CD47-Fc are in progress.
Antibodies against ROS-producing ragweed pollen proteins may prevent allergic sensitization (P6375)
Authors: Pazmandi Kitti , Kumar Brahma , Boldogh Istvan , Kemeny-Beke Adam , Szilasi Maria , Rajnavolgyi Eva , Bacsi Attila
Published: January 2023

We have previously described that hydrated pollen grains and their allergenic extracts possess intrinsic NAD(P)H oxidase activity, which catalyzes the production of reactive oxygen species (ROS) and induces oxidative stress in the lung or conjunctiva. This oxidative insult promotes the development of adaptive immune responses against innocuous pollen proteins and augments allergic airway inflammation induced by pollen antigens. In this study we investigated the ROS-producing activity of short ragweed (Ambrosia artemisiifolia) pollen extract (RWE) in detail. First, we found that in the presence of NADPH, RWE produces more ROS than after addition of NADH. An in situ nitroblue tetrazolium assay demonstrated that at least two distinct enzymes are responsible for ROS generation in RWE. The first one can use both NADPH and NADH as substrates, while the second one produces ROS only in the presence of NADPH. We observed that despite the fact that sera from non-allergic individuals have lower non-enzymatic antioxidant capacity, they exhibit higher efficacy in inhibition of ROS generation by RWE compared to sera from allergic patients. Western blot analysis revealed that antibodies against some pollen proteins associated with ROS production of RWE are present only in the sera of non-allergic subjects. Our novel findings raise the possibility that these antibodies may be able to block the ROS production by pollen enzymes hereby prevent the sensitization of non-allergic individuals.
Helicobacter pylori-derived heat shock protein 60 increases the induction of regulatory T cells (P3365)
Authors: Hsu Wei-Tung , Wu Ming-Shiang , Wu Chang-Jer , Chen Chia-Hung , Jian Ting-Yan , Liao Kuang-Wen
Published: January 2023

Regulatory T cells have been shown to highly infiltrate the H. pylori-infected gastric mucosa. Treg cells suppress the host’s immune responses to H. pylori and dominate during persistent infection. H. pylori-derived HSP60 is one important virulence factor that promotes malignant tumorigenesis. This study further investigated the role of HpHSP60 in immunosuppression, and its activity was found to correlate with the induction of Treg cells. To determine the effects of HpHSP60 on immunosuppression, human peripheral blood mononuclear cells were treated with or without HpHSP60 in the presence of anti-CD3 mAb to determine the effect of HpHSP60 on proliferation. We subsequently asked whether proliferative inhibition correlates with the induction of Treg cells. Finally, the mechanism through which HpHSP60 induces Treg cells was identified. HpHSP60 decreased the expression of CDK4 to significantly slow the proliferation of mitogen-stimulated T cells, which correlated with the induction of Treg cells. Moreover, secretion of IL-10 and TGF-β by monocytic cells was essential to the induction of HpHSP60-induced Treg cells. In summary, we propose that HpHSP60 can act on macrophages to trigger the expression of IL-10 and TGF-β, resulting in an increase in Treg cells and the proliferative inhibition of T cells.
Analysis of bronchoalveolar immune cells and cytokines induced by immunization with different respiratory syncytial virus vaccines (P4395)
Authors: Ko Eun-Ju , Lee Jong Seok , Lee Yu-Na , Kwon Young-Man , Hwang Hye Suk , Yoo Si-Eun , Quan Fu-Shi , Lee Sujin , Moore Martin , Kang Sang-Moo
Published: January 2023

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection and deaths in infants. Since the failure of formalin-inactivated RSV (FI-RSV) due to vaccine-enhanced pulmonary eosinophilia and pneumonia during 1960s, there is no licensed vaccine against RSV. In this study, we analyzed immune cell phenotypes and cytokines in bronchoalveolar lavage fluids (BALF) from mice immunized with FI-RSV, live RSV, or F plasmid and virus-like particles (VLP) containing RSV F and G glycoproteins (FFG VLP) at day 5 post RSV challenge. FI-RSV immunized mice showed severe eosinophilia, high levels of CD11b+ cells as well as IFN-gamma and IL-4 secreting CD4 T cells locally and systemically. Mice pre-immunized with live RSV showed no severe pulmonary eosinophilia, similar levels of IFN-gamma secreting CD4 and CD8 T cells, and low levels of IL-4 secreting cells. Mice immunized with FFG VLPs displayed no eosinophilia, relatively a low ratio of CD4/CD8 T cells secreting IFN-gamma, and no systemic cytokines. All 3 types of RSV vaccines were effective in clearing lung viral loads. Taken together, these results suggest no correlation between lung RSV clearance and inflammatory pulmonary disease, and that FFG VLP can be a promising RSV vaccine candidate conferring protection without lung disease.
Scavenger receptor SRECI can bind to Toll like receptor 4 and activate LPS-mediated signaling (P1362)
Authors: Murshid Ayesha , Gong Jianlin , Calderwood Stuart
Published: January 2023

Transmembrane receptor TLR4 was shown to induce the inflammatory response in the presence of its ligand, LPS. All TLRs belong to a family of PRR (pattern recognition receptor) which recognize PAMPs (pathogen-associated molecular patterns). The other receptor families, scavenger receptors work along with TLRs to attenuate innate immunity. SREC-I is a scavenger receptor expressed by immune cells and has a role in CD8+ T cell immunity. SREC-I can also modulate the function of Toll like receptors which have essential roles in innate immunity. Here we show that SREC-I can activate LPS mediated NFkB signaling. SREC-I can bind to TLR4 in the presence of LPS. We found that human monocyte/macrophage THP1 cells as well as bone marrow derived macrophages from mice exhibited increased responses to LPS and enhanced production of inflammatory cytokines such as IL-8, IL-6 with overexpressed SREC-I. We were able to show that downstream activation of NFkB requires either MyD88 or Trif adaptor protein. Our data suggested that SRECI is a cell surface receptor capable of internalizing LPS along with TLR4 as well as activating TLR4 mediated downstream cytokine production in macrophages.
Hyperglycemia triggers epigenetic machinery to enhance MyD88 expression in macrophages from type 1 diabetes mice (P1350)
Authors: Filgueiras Luciano , Sisti Flavia , Ferreira Ana , Wang Soujuan , Wang Zhuo , Jancar Sonia , Serezani C. Henrique
Published: January 2023

Type I diabetes (T1DM) is characterized by hyperglycemia and insulin deficiency. T1DM is associated with constitutive production of pro-inflammatory cytokine such as the members of IL-1 family. IL-1β binding to its MyD88-associated receptor IL-1R is a major cause of tissue injury in T1DM. We hypothesize that hyperglycemia (HG) enhances MyD88 in macrophages (MA) amplifying IL-1βR responsiveness. We found that both in vitro and in vivo HG increases the expression of MyD88 and its transcription factor STAT-1. Speculating that enhanced MyD88/STAT-1 levels could be due to epigenetic changes, we analyzed the expression of enzymes involved in DNA methylation (DNMT) and Histone deacetylation (HDAC) in MA from T1DM mice. We observed that diabetic mice expressed at least 2 times more HDAC 8, 10 and 11, and DNMT 3b than nondiabetic mice. In vitro inhibition of HDAC (TSA) or DNMT (5-azacyd) in MA impaired HG-enhanced MyD88 and STAT-1 expression. These data suggest a heretofore unrecognized epigenetic changes associated with hyperinflammatory pattern of MA from T1DM, leading to high MyD88 expression and possibly allowing overly IL-1β receptor and other MyD88-dependent receptor activation
Reactive oxygen probes - a broad range of colors with easier labeling and compatibility with fixation: novel CellROX® reagents from Molecular Probes® (P3295)
Authors: Bone Bradley , Seredick Barbara , Olszowy Mike
Published: January 2023

A natural consequence of aerobic respiration is the generation of highly toxic radicals called reactive oxygen species (ROS). ROS have been implicated in various human pathologies. Although fluorogenic probes such as Aminophenyl fluorescein (APF) and hydroxyphenyl fluorescein (HPF), and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) have been widely used to detect ROS using flow cytometry and imaging platforms, all are excitable by the 488nm laser and emit in the fluorescein channel, and require that labeling occur in a protein-free buffer. In contrast, the novel CellROX® ROS detection reagents from Molecular Probes® offer increased flexibility for multiplex experiments, with fluorescence emission in the fluorescein, PE, or APC channels for CellROX® Green, Orange, and Deep Red. In addition, the CellROX® reagents offer increased ease of use with the ability to label cells in complete growth media, have increased photostability as compared to H2DCFDA, and are compatible with fixation (CellROX® Green and Deep Red). The CellROX® ROS detection reagents are bright and stable ROS sensors that offer significant advantages over existing ROS sensors because they are compatible with labeling in different media and can be used with fixatives. For Research Use Only. Not for use in diagnostic procedures.
A single laser flow cytometer: simplifying multi-color immunophenotyping. (P3303)
Authors: Rohrer Jurg , Duckett Larry , Gaylord Brent , Stall Alan , Nguyen Andrea , Vidal Jason
Published: January 2023

Flow cytometry has been an indispensable tool for immunologists, clinicians and cell biologists for almost 40 years. The power of flow cytometry is in its ability to simultaneously measure multiple features (parameters) per cell and this unique aspect has formed the basis for most cell analysis applications to date. Initially cytometers were only able to distinguish 2 fluorescent dyes (or colors) but the technology has since advanced to multi-laser systems capable of up to 18 color detection. However, these instruments can be considered complex and costly for simple immunophenotyping experiments. The BD Accuri C6 was able to address this need as a simple to use instrument that could perform 4 color immunophenotyping on an individual’s lab bench as opposed to relying on core facilities. We have taken this concept a step further leveraging the exceptionally bright polymeric Sirigen dyes. These dyes, excited off the violet laser, span the visible to near IR spectrum giving one the opportunity to perform up to four color immunophentyping with minimal compensation while maintaining good signal resolution. In support of this, we will show data for simple T cell, B cell and NK cell immunophenotyping using a customized single violet laser Accuri. Details on optimizing filter sets will be given as well as details on how to optimize the reagent panels to be used with this laser/filter set up.
Apoptotic cell recognition receptors, Tyro3, Axl and Mer, demonstrate distinct patterns of ligand-inducible and ligand-independent receptor activation (P2016)
Authors: Tsou Wen-I , Nguyen Khanh Quynh , Birge Raymond , Kotenko Sergei
Published: January 2023

Proper recognition and removal of apoptotic cells by phagocytes lead to the active suppression of inflammatory responses and the induction of tolerance. Tyro3, Axl and Mer (TAM) receptor tyrosine kinases recognize apoptotic cells through their ligands, Protein S (ProS) and Growth-Arrest-Specific Gene 6 (GAS6), which in turn interact with phosphatidylserine (PS) exposed on the surface of apoptotic cells. Although TAMs share significant similarity, very little is known about the specificity of interaction between TAMs and their ligands, in the context of apoptotic cells, and about downstream signaling cascades triggered through TAMs. To study ligand-receptor interaction, we generated a series of reporter cell lines expressing chimeric TAM receptors that allowed us to demonstrate that each TAM has a unique pattern of interaction with GAS6 and ProS, which is also differentially affected by the presence of apoptotic cells. We also developed another set of chimeric receptors that triggered ligand-independent activation of the TAM intracellular domains, leading to distinct patterns of phosphorylated signaling molecules, as well as different levels of cytokine induction. Overall, these studies suggest that despite their similarity, Tyro3, Axl and Mer are functionally unique in the recognition of apoptotic cells, activation of downstream signaling cascades and gene expression, and therefore, contribute differently to cell survival and the induction of tolerance during tumorigenesis.
Antibodies of a novel bunyavirus in domestic and captive farmed animals in the Midwestern United States (P6371)
Authors: Xing Zheng , Schefers Jeremy , Schwabenlander Marc , Jiao Yongjun , Liang Mifang , Qi Xian , Li Chuan , Goyal Sagar , Cardona Carol , Li Dexin , Collins James , Murtaugh Michael
Published: January 2023

The Heartland virus (HLV), a new phlebovirus isolated in Missouri from patients with a history of tick bites and symptoms similar to severe fever with thrombocytopenia syndrome (SFTS), is an emerging pathogen. Genetically HLV is closely related to the newly identified bunyavirus (SFTSV) causing SFTS. In this study we tried to identify animal hosts that may be susceptible to and infected with HLV, or a SFTSV-like virus, and may serve as amplifying hosts important in virus transmission in the United States. Blood samples were obtained from several domestic and captive farmed animal species from 29 counties of Minnesota in 2012. An antigen sandwich ELISA for detection of SFTSV NP antibodies was used. Antibodies to SFTSV NP were detected in sera from 64 of 414 cattle (15.5%), 10 of 92 goats (10.9%), 6 of 48 sheep (12.5%), 35 of 296 white-tailed deer (11.8%), and 7 of 39 elk (18.0%). Thirty-four of 64 positive samples had P/N ratios between 4 and 10; and 11 had ratios ≥10. Our data show that both domestic and captive farmed animals were exposed to SFTSV- or HLV-like virus in the Midwestern United States. Since SFTSV and HLV are closely related, the viruses detected in the Midwestern region are most likely HLV or close relatives of HLV. Evidence that a novel phlebovirus infects a variety of domesticated and captive farmed animals as shown in this study validates the concern that SFTSV or HLV could pose a serious threat to animal welfare and public health in the United States.
Characterization of CMV- and LCV-specific T cell responses and their sensitivity to immunosuppression in cynomolgus macaques (P6367)
Authors: Kamperschroer Cris , O'Donnell Lynn , Schneider Patricia , Li Dingzhou , Kawabata Thomas
Published: January 2023

Herpesviruses such as cytomegalovirus (CMV) and Epstein Barr virus (EBV) often cause disease under conditions of immunosuppression, such as transplant or AIDS. Loss of control of herpesvirus infections occurs at least in part due to loss or dysfunction of virus-specific T cells. An increasing array of immunosuppressive therapeutics are being developed that target different subsets of T cells or their functions. To better understand effects of therapeutics on antiviral T cells, we have used ELISPOT and ICS to measure T cell responses against CMV and lymphcryptovirus (LCV; analogous to EBV) in cynomolgus macaques. Our work has shown that nearly all monkeys respond against both viruses, although the numbers of T cells specific for CMV are larger than those against LCV. We have also characterized the variability in the magnitude of the T responses to both viruses over time. In addition, we have examined the effects of immunosuppressive agents on the numbers and function of antiviral T cells. Our work reveals remarkable stability of herpesvirus-specific T cells, even under conditions where drugs abolish T cell stimulation for a period of several weeks.
LAMP-2C regulation of MHC class II antigen presentation (P5020)
Authors: Pérez Liliana , Blum Janice
Published: January 2023

Chaperone-mediated autophagy (CMA) is a selective autophagy pathway, which relies on the lysosome-associated membrane protein (LAMP)-2A to translocate proteins from the cytoplasm into lysosomes. Alternative splicing of the LAMP-2 gene generates 3 highly conserved isoforms LAMP-2A, LAMP-2B and LAMP-2C. LAMP-2A overexpression increases MHC class II (MHCII) presentation of the cytoplasmic autoantigen, GAD, demonstrating a role for CMA in antigen presentation. Overexpression of LAMP-2B did not alter cytoplasmic GAD presentation, however, remarkably ectopic expression of LAMP-2C in B cells inhibited T cell responses to cytoplasmic GAD. Studies were undertaken here to understand the role of LAMP-2C in MHCII antigen presentation. The presentation of several exogenous proteins was not affected by LAMP-2C expression in B cells. Similar results were observed for the presentation of endogenous self and viral membrane proteins. These results suggest that LAMP-2C may be a specific inhibitor of antigen presentation via autophagy. Ectopic expression of LAMP-2C also failed to perturb MHCII presentation of the MP1-LC3 antigen via macroautophagy. Thus LAMP-2C appears to inhibit the presentation of cytoplasmic proteins that are processed through CMA and may not affect other autophagy pathways. Future studies are underway to determine mechanistically how LAMP-2C controls antigen presentation.
Cross-presentation to CD8+ T cells is critical to maintain protective immunity against chronic phagosomal bacteria (P5030)
Authors: Patel Rajen , Sad Subash
Published: January 2023

Salmonella Typhimurium (ST) is a virulent intracellular pathogen that resides within the phagosomes of infected cells. Dendritic cells (DCs) are capable of presenting phagosomal antigen to prime CD8+ T cells through the cross-presentation pathway. However, the role of CD8+ T cells and cross-presentation in immunity against ST has yet to be fully elucidated. We utilized recombinant ST expressing ovalbumin to study cross-presentation of cell-associated antigen during infection. Mice depleted of CD8+ T cells exhibit higher bacterial burden and increased mortality. DCs isolated from Ifnαr-/- mice, deficient in the cross-presentation pathway, exhibit attenuated antigen presentation against ST. However, our in-vivo data suggests that IFN-I mediated cross-presentation of ST is dependent on the route of infection, as Ifnαr-/- mice exhibit defects in DC trafficking to lymph nodes. Furthermore, we observed IFN-I mediated CD8+ T cell response to be pathogen specific, as antigen presentation of Listeria monocytogenes, an acute intracellular pathogen, was not attenuated in Ifnαr-/- mice. Interestingly, Batf3-/- mice deficient in CD8α DCs, a subset of DCs that specialize in cross-presentation, results in a significantly delayed CD8+ T cell response against ST and higher bacterial burden. Collectively, our data indicates that cross-presentation is an essential mechanism to mount a CD8+ T cell response against ST and is necessary for maintaining immune protection against the bacterium.
Serum from patients with SLE instructs monocytes to promote auto-antigen specific IgG and IgA responses (P4028)
Authors: Joo HyeMee , Xue Yaming , Upchurch Katherine , Zurawski Gerard , Pascual Virginia , Oh SangKon
Published: January 2023

The persistent development of autoantibodies is a hallmark of systemic lupus erythematosus (SLE). Previous work using SLE serum demonstrated that monocyte differentiation into dendritic cells (DCs) is a type I IFN-dependent manner and such SLE-DCs activate T cells. However, whether SLE-DCs promote B cell responses has not been elucidated. Here, we demonstrate that SLE-DCs can efficiently stimulate naïve and memory B cells to differentiate into polyclonal IgG- and IgA-plasmablasts (PBs) resembling those found in the blood of SLE patients. More importantly, when SLE-DCs were stimulated with SLE neutrophils, these SLE-DCs were able to induce ss-DNA specific IgG and IgA from naïve B cells. SLE-DC-mediated IgG-PB differentiation is dependent on BAFF and IL-10, whereas IgA-PB differentiation is mainly dependent on APRIL. Moreover, SLE-DCs as well as monocytes from SLE patients express CD138 and thus can trans-present CD138-bound APRIL to B cells, which is crucial for inducing IgA switching and PB differentiation. The expression of CD138-bound APRIL is not dependent of IFNα, explaining why DCs generated with IFNα (IFN-DCs) are less efficient at inducing IgA responses. Nonetheless, both SLE-DCs and IFN-DCs induce the expression of Bcl-XL in PBs and support their survival, thereby leading to increased Ig production. Taken together, our study suggests that a direct interplay between SLE-DCs and peripheral B cells contribute to SLE pathogenic autoantibody responses.