bluuebunny/crossref_metadata_2025 huggingface dataset:
Pattern of cytokine response to Mycobacterium leprae following interruption of persistent exposure to leprosy and BCG vaccination (MPF6P.653)
Authors: de Carvalho Fernanda , Rodrigues Luciana , Alvim Iris Maria , Alves Marcelo , Pinheiro Roberta , Duppre Nádia , Sarno Euzenir , Pereira Geraldo , Pessolani Maria Cristina
Published: January 2023

In 1973 Godal and collaborators investigated the in vitro response to M. leprae in leprosy patients and their household contacts. The authors observed a defective proliferative response to ML in lepromatous leprosy patients (LP), and also a reduced responsiveness specific for M. leprae, in household contacts of lepromatous leprosy patients. The responsiveness of these contacts to M. leprae improved upon treatment of the lepromatous patients (index cases). In the present study we assessed the impact of BCG vaccination and the leprosy index case treatment on the in vitro pattern of response to ML of household contacts of multibacillary leprosy patients (HCMB). Blood leukocytes of HCMB (n=16) were isolated at the beginning (T0) and during the course of the leprosy index case treatment (at least six month of treatment) (T1). At T0 thirteen HCMB were vaccinated with BCG. Most of the HCMB showed increased frequency of central memory and effector memory CD69+CD4+ T cells in response to M. leprae and peptides at T1. The evaluation of ratios of T1/T0 cytokine responses, using principal component analysis, indicates that in response to M. leprae the cytokines IL-6, IL1β, TNF-α, IL-17 and IFN-γ increased following interruption of exposure to ML, and BCG vaccination. Thus , the persistent exposure to ML may facilitate evolution of infection to active disease by inhibiting the effector response against ML.
Bifidobacterium longum strain change CD4 and CD8 T cells profile in inflammatory bowel disease induced in mice (MPF6P.663)
Authors: Wasilewska Ewa , Zlotkowska Dagmara
Published: January 2023

Probiotics are defined as a live microorganisms which, when given to the host, beneficially affect the properties of the natural microflora. In our earlier study, we found reduction of the amount of CD8+ and CD4+ T cells in PP in mice fed with different commercial probiotic preparations. Nevertheless, some of them significantly induced CD8+CD4+ cells, most effectively enhanced CD4+ and lowered CD8+ cells in splenocytes, but later, this activating effect was reduced and changed. The presented study compared the effect of potentially probiotic Bifidobacterium longum strain on DSS - induced colitis in older mice. Colitis was induced for 7 days via administration of 2.5% DSS in drinking water. BALB/c mice were gavaged 200 µl of the probiotic preparation (5x109 CFU/mL) or physiological saline, for 14 days. Feeding mice with B. longum significantly increased CD4+ and decreased CD4+CD8+ T cells in MLNs. Significant reduction of CD4+CD8+ T cells was also observed in PPs, however, with no changes in the other populations tested. Splenocytes responded to bacterial feeding by significant increasing both CD4+ and CD8+ T cells, whereas, lymphocytes from peripheral lymph nodes respond by further increase of CD4+, but decrease of CD8+ cells. No changes in lymphocytes phenotyping in the two tested groups were observed in the serum. The experiment showed that the strain tested changed T lymphocyte profile, and further studies are necessary to evaluate its safety and effectiveness.
Protective IL-10-producing Th1 cells also make IL-21, which is regulated by the T-bet/Bcl6 ratio, in a mouse model of chronic malaria (MPF7P.709)
Authors: Carpio Victor , Opata Michael , Montañez Marelle , Dent Alexander , Banerjee Pinaki , Stephens Robin
Published: January 2023

Chronic exposure to the malaria parasite is required for full clinical protection, possibly due to improved survival versus maintenance of effector function of CD4 memory T cells (Tmem). IFN-γ is required for protection; therefore, we investigated the development of Th1 memory in chronic Plasmodium chabaudi malaria infection using ifng-reporter mice. We observed that the expression of Th1 markers (T-bet, CXCR3) peaked well before T cell contraction, suggesting poor long-term commitment to IFN- γ production. In addition, markers of T follicular helper cells (Tfh), such as CXCR5 and Bcl6, were on most Th1 cells, and some co-produced IL-21 and IL-10. While Th1 effector cells differentiate into CXCR3+ Tmem in malaria, they are Tbetlo and do not maintain ifng expression, unless they are dividing, though they do express Bcl6 and IL-21. Interestingly, Bcl6-deficient Th1 cells produce more IL-21, while infection of IL-10-deficient mice increased T cell expression of both T-bet and Bcl6, generating more mixed phenotype cells. P. chabaudi infected IL-10-/- mice die of T cell-mediated pathological inflammation, suggesting that the dominant Th1/Tfh phenotype is pathogenic if not regulated. Expression of Bcl6 and IL-21 in Th1 cells has been described as an early and transient event before full commitment; however, these results suggest that this intermediate stage can be maintained in chronic infection, potentially to prevent both exhaustion and pathology, via IL-10.
Role of interleukin-33 during Schistosoma mansoni infection (MPF7P.713)
Authors: Kalenda Yombo , Masuda Kyoko , Sayed Eman , Nakamura Risa , Kato Kentaro , Uematsu Satoshi , Akira Shizuo , Hamano Shinjiro
Published: January 2023

The host immune response to Schistosoma infection is characterized by two main phases, Th1 and Th2 consecutively, the later one being related to the excretion of mature eggs by the adult female worm. Interleukin-33 (IL-33), a Th2 inducing cytokine, has been reported to be associated with schistosomiasis. Here we assessed the role of this epithelial related cytokine on the time course of the experimental S. mansoni infection in mouse. IL-33 knockout (KO) and wild type (WT) 6-week-old BALB/c female mice were infected with 50 cercariae and sacrificed at 0, 6, 8 and 10 weeks post infection. We monitored symptoms of mice, the worms and eggs burden, kinetics of immune cells in the liver and spleen, and cytokine levels. The size of granuloma induced in the liver and intestine were also measured. There was no significant difference between KO and WT mice in the survival rate, worms and eggs burden; nevertheless some sparse differences related to the increase of eosinophils were detected at 8 weeks post-infection. A very few number of KO mice displayed a severe anemia and colonic bleeding at the end of the experiment, suggesting some intestinal effects of IL-33 as epithelial alarmin. Our results suggest that although IL-33, the Th2 inducing cytokine, was reported to be crucial in other helminthic diseases, it may have little impact on the course of S. mansoni infection.
Inhibiting the mammalian target of rapamycin as an adjunctive therapy for cerebral malaria (MPF7P.714)
Authors: Hart Geoffrey , Gordon Emile , Waisberg Michael , Tran Tuan , Akkaya Munir , Skinner Jeffrey , Zinöcker Severin , Pena Mirna , Yazew Takele , QI chenfeng , Miller Louis , Pierce Susan
Published: January 2023

Malaria is an infectious disease caused by parasites of the Plasmodium spp. In endemic areas, in approximately 1% of cases, almost exclusively in young children, malaria becomes severe resulting in nearly seven hundred thousand deaths each year in Africa alone. Cerebral malaria (CM) is a common form of severe malaria and one for which we have no effective adjunctive therapy. The kinase, mammalian target of rapamycin (mTOR), through its regulation of cellular metabolism, is a central regulator of immune responses and drugs that inhibit the mTOR pathway have been shown to be antiparasitic, raising the possibility that mTOR inhibitors could be effective adjunctive therapies for CM. Here we show in a mouse model of CM, experimental CM (ECM), that the mTOR inhibitor rapamycin protects against ECM when administered within the first four days of infection. Treatment with rapamycin increased survival, blocked breakdown of the blood brain barrier and brain hemorrhaging, decreased influx of both CD4+ and CD8+ T cells into the brain and the accumulation of parasitized red blood cells in the brain. Remarkably, animals were protected against ECM even though rapamycin treatment significantly increased the inflammatory response induced by infection in both the brain and spleen and elevated the levels of peripheral parasitemia. These results open a new avenue for the development of highly selective adjunctive therapies for CM, by targeting pathways that regulate host and parasite metabolism.
IL-27 signaling is crucial for survival of mice infected with African trypanosomes via preventing lethal effects of CD4+ T cells and IFN-γ (MPF7P.719)
Authors: Liu Gongguan , Xu Jinjun , Sun Donglei , Shi Meiqing
Published: January 2023

African trypanosomes are extracellular protozoan parasites causing a chronic debilitating disease with a persistent inflammatory response. IL-10 signaling is essential to maintain the balance of the inflammatory response. Here we demonstrate that IL-27R-deficient (WSX-1-/-) mice are highly susceptible to infection with Trypanosoma congolense and T. brucei, developing exacerbated Th1-mediated immune responses with highly activation of CD4+ T cells and strongly enhanced production of inflammatory cytokines including IFN-γ, which result in severe liver immunopathology and dramatically reduced survival. IL-10 signaling was not impaired in infected WSX-1-/- mice. Either depletion of CD4+ T cells, but not CD8+ T cells, accompanied by a significantly reduced production of IFN-γ, or neutralization of IFN-γ, prevented the early mortality of infected WSX-1-/- mice with a significantly reduced inflammatory response and a major amelioration of liver pathology, providing direct evidence that CD4+ T cells mediated the early mortality of infected WSX-1-/- mice directly via excessive secretion of IFN-γ. Thus, our data identify IL-27 signaling, in addition to known IL-10 signaling for limiting M1-type myeloid cells, as a novel pathway to prevent early mortality via inhibiting hyperactivation of Th1 cells during infection with African trypanosomes. These data are the first to demonstrate the essential role of IL-27 signaling in regulating immune responses to extracellular protozoan infections.
Metalloprotease NleC suppresses host NF-κB/inflammatory responses by cleaving p65 and interfering with the p65/RPS3 interaction (MPF7P.703)
Authors: Hodgson Andrea , Wier Eric , Fu Kai , Sun Xin , Zheng Wenxin , Sham Ho Pan , Yu Hongbing , Johnson Kaitlin , Bailey Scott , Vallance Bruce , Wan Fengyi
Published: January 2023

The NF-κB signaling pathway has long been recognized as a crucial activator and regulator of the innate and adaptive immune responses. More recently, the ribosomal protein, RPS3 has been identified as a non-rel component of NF-κB and functions as a “specifier” protein critical for the induction of selective proinflammatory gene transcription. Attaching/effacing pathogens including enteropathogenic Escherichia coli, enterohemorrhagic E. coli and the rodent equivalent Citrobacter rodentium are important causative agents of foodborne diseases. A/E pathogens have evolved many strategies to evade the host immune response and encode various effector proteins that target NF-κB at different stages. Our work here, demonstrates how the A/E encoded metalloprotease NleC, specifically cleaves the p65 molecule at the N-terminus generating a fragment that is capable of selectively blocking the function of RPS3. This selective blockade of RPS3 results in a dampened immune response following infection, which amplifies the effect of cleaving only a small percentage of p65 to modulate NF-κB-mediated gene expression. Thus, our results reveal a novel mechanism for A/E pathogens to specifically block NF-κB signaling and inflammatory responses by cleaving a small percentage of p65 and targeting the p65/RPS3 interaction in host cells, thus providing novel insights into the pathogenic mechanisms of foodborne diseases.
Vanilloid receptor 1 acts as a regulator of immune homeostasis in the gut (MUC8P.731)
Authors: Acharya Nandini
Published: January 2023

Endogenous cannabinoids (endocannabinoids) are small molecules biosynthesized from membrane glycerophospholipids. Endogenous intestinal cannabinoids such as Anandamide, control appetite and energy balance by engagement of the enteric nervous system through cannabinoid receptors. Here, we show that Anandamide and its vanilloid receptor, VR1, mediate immune homeostasis in the gut and pancreas. Capsaicin, a vanilloid, with which Anandamide shares VR1, acts similarly. Engagement of the cannabinoid/vanilloid receptor augments the number and immune suppressive function of the regulatory CX3CR1hi macrophages, which express the highest levels of such receptors among the gut immune cells. Additionally, mice that are genetically deficient in VR1 have reduced proportions of CX3CR1hi macrophages in the gut. Treatment of mice with capsaicin also leads to differentiation of a regulatory subset of CD4+ T cells called Tr1 cells in an IL27 dependent manner both in vitro and in vivo. In a functional demonstration, tolerance elicited by engagement of VR1 can be transferred to naïve NOD mice (model of type 1 diabetes) by transfer of CD4+ T cells. Our study unveils a novel role for cannabinoids in maintaining immune homeostasis in the gut/pancreas, and reveals a new conversation between the nervous and immune systems, using distinct receptors.
Chronic ethanol consumption alters intestinal microbiota composition (MUC9P.746)
Authors: Barr Tasha , George Michael , Grant Kathleen , Messaoudi Ilhem
Published: January 2023

It is well known that chronic alcohol consumption leads to organ damage and increased susceptibility to bacterial and viral infections. Ethanol exposure in the intestinal lumen alters tight junction proteins leading to disruption of the epithelial barrier. Increased permeability in turn leads to translocation of gut-derived endotoxins causing inflammatory responses in the liver that contribute to alcoholic liver disease. Recent studies have shown that gut mucosa homeostasis is also modulated by the microbiome. Disruptions in the gut microbiome have been described for many diseases including IBD, diabetes and colon cancer. In order to gain a better understanding of the dose-dependent effects of ethanol on the gut microbiome, we performed ribosomal RNA sequencing to determine the differentially expressed bacteria in the small and large intestine in a rhesus macaque model of voluntary ethanol self-administration. In the colon we observed a 2-fold increase in heavy drinkers compared to nondrinkers in the phylum Bacteroidetes that subsequently results in a decrease in Firmicutes, the phylum containing most beneficial lactic acid bacteria. In the duodenum, jejunum, ileum and colon we found a 3-fold decrease in the commensal species Lactobacillus intestinalis and a 2-fold increase in the potential pathogenic species Prevotella copri in heavy drinkers compared to nondrinkers. These studies are likely to reveal mechanisms of ethanol-related gut dysregulation and related inflammation.
Quantitation of activating signaling microclusters’ size and dynamics and their influence on killing kinetics by cytotoxic lymphocytes (TECH2P.915)
Authors: Sykulev Yuri , Steblyanko Maria , Anikeeva Nadia , Keen James
Published: January 2023

Stimulation of cytotoxic lymphocytes via activating receptors results in aggregation of the receptors and recruitment of proximal signaling proteins at distinct location of the contact surface term signaling microclusters. The microclusters that are initially arise in the periphery of the contact surface then move centripetally to the center and signal when they are on the move. We examined the size and dynamics of CD16 activating signaling microclusters at the interface of CD16.NK-92 cells and supported lipid bilayers containing anti-CD16 antibody and ICAM-1, a ligand for integrin receptors, at different densities. We have found that variations in the ligation of integrins but not CD16 receptor significantly affect the microclusters’ size and their displacement and mobility. These and other data provided evidence that integrin signaling effectively modulate activating receptor proximal signaling and regulate the kinetics of cytolytic granule delivery and killing kinetics by cytotoxic lymphocytes.
Single cell gene expression in lupus patient monocytes reveals a differential impact of interferon signaling between monocyte subtypes (TECH2P.920)
Authors: Niewold Timothy , Jensen Mark , Dorschner Jessica , Vsetecka Danielle , Amin Shreyasee , Makol Ashima , Ernste Floranne , Osborn Thomas , Moder Kevin , Chowdhary Vaidehi , Jin Zhongbo
Published: January 2023

Background/Purpose Interferon (IFN) gene expression signatures have been observed in whole blood and mixed immune cell populations in systemic lupus erythematosus (SLE), but the significance of this IFN signaling in immune cell subsets is not well understood. We examined gene expression in individual SLE patient monocytes to explore the impact of increased IFN-induced transcription in single cells. Methods CD14++CD16- classical monocytes and CD14dimCD16+ non classical monocytes from SLE patients were purified by magnetic separation. Single cells were isolated, and gene expression in each individual cell was examined by rtPCR. Results Monocytes from the same SLE patient blood sample demonstrated varying levels of IFN-induced gene expression. In classical monocytes, high IFN score correlated with CD32a, IL1B, and IL8 expression. In non-classical monocytes, high IFN score was correlated with a larger number of inflammatory mediators, including cytokines such as IL12, IL23, and IL15; the immune receptors CD36, CD32a, CD80, and TLR7; and inflammatory signaling genes such as RELA, STAT2, IRAK1, IRAK4, and MyD88. Conclusion We observed striking diversity in the IFN responses of individual monocytes, demonstrating that IFN signaling has distinct effects upon classical and non-classical monocytes. Single cell studies can reveal effects of IFN on single immune cells and uncommon populations such as non-classical monocytes, which would be masked in whole blood or mixed cell populations.
Profiling a structure activity relationship library for immunological activity in human PBMCs: multiplexed measurements of cell phenotype, viability, and cytokine secretion from a single high throughput screen (TECH2P.923)
Authors: Duensing Thomas , Liu Zhaoping , Brodbeck Daniela , Parel Serge , Luu Yen
Published: January 2023

The success of immunomodulatory approaches for the treatment of disease has generated tremendous interest in discovery of new immunotherapy based therapeutics. Immunotherapy has the potential of affecting - positively and negatively - the myriad interactions among cells and signaling molecules regulating the immune system, and projects should include high throughput screening campaigns aimed at profiling the effects of compounds on these complex interactions. We describe a no-wash, high throughput screen that provides a multiparameter activity profile for a structure activity relationship (SAR) library of immunomodulatory compounds. Seven known immunomodulatory compounds were used as templates to generate an SAR expansion library of 1438 compounds. The selection utilized a combination of various substructure and similarity search methods combined with the generation of virtual templates. Phytohemagglutinin (PHA) activated PBMCs were treated with the compounds at two doses, 10µM and 50µM. Immunophenotyping was done by α-CD3 and α-CD8 antibodies and differential toxic effects of compounds on the viability of individual cell subsets plus effects on the secretion of three cytokines were used to generate an immunological profile for each compound tested. We demonstrate the combination of a robust SAR expansion system with high throughput, multiplex screening on primary cells to generate activity profiles that can be used for identifying viable therapeutic candidates.
Whole blood fixation and cryopreservation procedures allow cellular analysis by flow cytometry for up to 30 days after cell isolation (TECH2P.763)
Authors: Paredes R. , Dalle Lucca Jurandir , Crossland Randy , Sheppard Forest
Published: January 2023

Background: Pre-clinical and clinical immunotyping of whole blood (WB) by flow cytometry (FC) is commonly used to assess immune system changes. Current recommendations require FC analysis of pre-stained samples to be completed within 24 h, imposing logistical constraints. Development of sample preservation methods for FC is crucial for extending this technology to locations not equipped with FC and high-throughput studies. Here, we tested a sample preservation method to extend the time window for FC analysis. Methods: Paired fresh WB samples from 4 Rhesus macaques were untreated or stimulated (100 µg/ml LPS, 37oC, 22 h), surface stained, fixed, permeabilized, and intracellular (IC) stained immediately or after preservation by freezing (-20oC; 5, 7, 15, 30 days). Mean fluorescence intensity (MFI) was quantified (BD FACSAria II). Statistical analysis between frozen and fresh samples was performed by the Friedman test with significance at p≤0.05. Results: There were no significant changes over 30 d in MFI from surface markers (CD3, p=0.69; CD4, p=0.32; CD8, p=0.11) or IC cytokines in either lymphoid (IL-8, p=0.98; IL-10, p=0.33; TNFα, p=0.32) or myeloid populations (IL-8, p=0.76; IL-10, p=0.23; TNFα, p=0.14). Conclusion: In frozen WB cells, FC revealed surface marker fluorescence can be maintained for up to 30 days with stable staining of IC markers. These findings have implications for the use of FC in locations with resource constraints, as well as in high-throughput analysis.
RNA fluorescence in situ hybridization and flow cytometry (FISH-Flow) detects antigen-specific T cell responses associated with latent tuberculosis infection (TECH3P.940)
Authors: Vir Pooja , Arrigucci Riccardo , Lakehal Karim , Pine Richard , Bushkin Yuri , Tyagi Sanjay , Davidow Amy , Lardizabal Alfred , Gennaro Maria
Published: January 2023

We have previously described fluorescence in situ hybridization for mRNA detection combined with flow cytometry (FISH-Flow) as a novel method to detect gene expression at a single-cell level. Using mRNA as analyte in flow cytometry presents multiple, biological and technical advantages. Proof-of-principle experiments have also shown that FISH-Flow can detect rare events including antigen-specific T cell responses. Whether the method is applicable to a biomedical problem remains to be determined. Here we applied FISH-Flow to detection of latent Mycobacterium tuberculosis infection (LTBI), a condition affecting approximately a third of the world population. LTBI diagnosis is based on measuring IFN-γ release from peripheral blood mononuclear cells (PBMC) stimulated ex vivo with M. tuberculosis antigen. We obtained PBMC from 60 donors equally distributed between LTBI+ and LTBI- individuals, stimulated them for 6 hrs with M. tuberculosis purified protein derivative (PPD), and measured expression of IFNG and IL2 mRNA by FISH-Flow. The proportions of antigen-induced IFNG and IL2 expression in CD3+ cells were >2-fold higher in LTBI+ than in LTBI- donors (p<0.005). Detection of multiple surface protein markers in combination with mRNA probes determined that CD4+ T cells are the primary cytokine-expressing subset. These results show that the FISH-Flow assay can identify a biological response and demonstrates the usefulness of this technique for basic and clinical immunological studies.
New multiplex interferon panel for simultaneous quantification of 13 human cytokines involved in anti-virus responses (TECH3P.934)
Authors: Sun Binggang , Lopes Patricia , Ji Shaoquan
Published: January 2023

In response to pathogens, especially viruses, cells release interferons and other cytokines to fight the infections. Interferons are typically divided into three types: I (e.g., INF-α, IFN-β), II (e.g., IFN-γ), and III (e.g., IFN-λ1, IFN-λ2). All interferons are important for fighting viral infections and for regulating the immune system. In addition, interferons are critically involved in cancer and autoimmune diseases such as psoriasis, systemic lupus erythematosus, and multiple sclerosis. Expression profiling of interferons and other related cytokines is critical in achieving a deeper understanding of the immune responses and various disease processes. We have developed a multiplex Human Interferon Panel, using fluorescence-encoded beads that are suitable for use on various flow cytometers. This panel allows simultaneous quantification of 13 cytokines involved in anti-virus responses including interferons (α, β, γ, λ1 and λ2), interleukins (1β, 6, 8, 10, 12), TNF-α, IP-10 and GM-CSF. Each antibody pair was carefully selected for assay specificity, sensitivity, accuracy and reproducibility. The panel has been validated by detecting expected changes in biological samples. Further advantages include high sensitivity, small sample volume, flexible assay configurations, and time- and cost-effectiveness. The Interferon Panel can be used for serum, plasma, cell culture supernatant and other sample types, offering a useful tool for biomedical research and drug discovery.
A 13-color parametric analysis for TBNK subset for the CytoFLEX instrument (TECH3P.935)
Authors: Lawson Jonel , Fischer Karen , Tung James
Published: January 2023

CytoFLEX is the newest cytometer offered by Beckman Coulter, Inc. that provides up to 3 lasers, 13 colors for detection in a compact system. This instrument incorporates the latest technology, Fiber array photodiode, FADP, to provide high resolution, dynamic range and sensitivity in signal detection. To demonstrate the ability of its multicolor detection, the standard multicolor panel design principles were followed to develop a 14 marker, 13 color experiment. The cocktail design process also took into account the desired immunophenotypic markers, the available fluorochromes, and the use of well-defined control populations to identify the classic TBNK populations. Data was collected from normal, whole blood stained with single color reagents and lysed with VersaLyseTM Lysing Solution. Data was analyzed using the CytExpert software and is presented here with special emphasis on the optimization of flow analysis and compensation visualization. Results show that all TBNK populations were well resolved, notably the CD25+ subset in CD4+ T-cells, even though this maker was placed in the PC7 channel, which normally has poor resolution. The data presented demonstrates the ability of the CytoFLEX to provide sound detection and resolution for even the most dim versus negative population.
Anti-inflammatory and neuroprotective effects of GILZ analogs. (THER7P.951)
Authors: Srinivasan Mythily , Chopra Nipun , Bayon Baindu , Lahiri Debomoy
Published: January 2023

Increased activation of the p65 subunit of nuclear factor kappa-B (NF-κB) upregulate inflammatory and apoptotic factors that mediate neuroinflammation and neurodegeneration in chronic neurological diseases, suggesting that activated p65 is an excellent therapeutic target. Glucocorticoid induced leucine zipper (GILZ) is a p65 interacting protein, that sequesters activated p65 in the cytoplasm and suppresses NF-κB. The GILZ:p65 interaction is localized to the proline rich region at the carboxy terminus of GILZ. Previously we reported that a synthetic peptide (GILZ-P) derived from the p65 binding domain of GILZ ameliorated experimental encephalomyelitis. To determine the potential application of GILZ-P in humans, we evaluated the toxicity and efficacy of the peptide drug in mature human neuroblastoma cells, macrophage like THP-1 cells and fetal neuronal cells. Treatment with GILZ-P at a wide range of concentrations commonly used for peptide drugs was non-toxic as determined by the cell viability and apoptosis assays in THP-1 cells. Functionally GILZ-P suppressed pro-inflammatory cytokine secretion by proteolipid protein or amyloid-β (Aβ1-42) stimulated mature neuroblastoma cells and suppressed glutamate secretion by activated macrophages by inhibiting nuclear translocation of p65. Collectively, GILZ-P exhibits therapeutic potential for chronic diseases wherein persistent inflammation precipitates neurodegeneration such as progressive multiple sclerosis and Alzheimer’s disease.
Arctigenin regulates Th1 and Th17 differentiation and ameliorates experimental autoimmune encephalomyelitis (THER7P.958)
Authors: Zhang Rongxin , Li Wen , Zhang Zhihui , Zhang Zimu , Xue Zhenyi , Li Yan , Zhang Lijuan , Da Yurong , Yao Zhi
Published: January 2023

Arctigenin is a phenylpropanoid dibenzylbutyrolactone lignan extracted from Arctium lappa (L.), and exhibit antitumor, antioxidant and neuron protective activities. But the effects of arctigenin on CNS inflammatory diseases are still unclear. Experimental autoimmune encephalomyelitis (EAE) is an animal model thought to be a T cell-mediated inflammatory demyelination disease. In this study, we in vivo explore the effect of arctigenin on mouse CD4+ effect T helper cell functions. Furthermore, in vitro explore the therapeutic effects and cellular mechanisms of arctigenin on EAE. Arctigenin treated mice are resistant to EAE, the clinical score of arctigenin treated mice is significantly reduced. Histochemistry assay of spinal cord sections also showed that arctigenin reduced inflammation and demyelination in EAE mice. The Th1 and Th17 cells were decreased in the spleens and lymph nodes of arctigenin treated EAE mice, while Th1 cells in CNS did not have significant changes. The expression of Th1 and Th17 cell proliferation and polarizing cytokines are inhibited in arctigenin treated mice spleens and lymph nodes. The Th1 cytokine IFN-γ and transcription factor T-bet, and Th17 cytokines IL-17A, IL-17F, and transcription factor ROR-γt, were significatly suppressed in vitro and in vivo. These suggest that arctigenin have properties of anti-inflammation and immunosuppression, which could be a potential therapeutic drug for multiple sclerosis and other autoimmune inflammatory diseases.
TNF regulated disability in experimental autoimmune encephalitis (THER7P.946)
Authors: Valentin-Torres Alice , Phares Timothy , Stohlman Stephen
Published: January 2023

In EAE, abrogation of IFN-γ signaling in astrocytes results in a progressive clinical disease, with increased CNS inflammation and demyelination, making this an excellent mouse model to study progressive multiple sclerosis (MS). Microglia and infiltrating macrophage TNF production is elevated during progressive EAE which is linked to pathology of several neurodegenerative disorders including MS. Moreover, anti-TNF therapies have been successfully used to treat several autoimmune diseases such as rheumatoid arthritis, Crohn’s disease, and psoriatic arthritis. Neutralization of both soluble and membrane bound TNF during progressive EAE significantly ameliorated disease progression and decreased demyelination. TNF blockade reduced the overall infiltration of immune cells into the CNS including IL-17 and IFN-γ producing T cells by restoring the blood brain barrier integrity. A reduction in the number of microglia and macrophages expressing MHC II+ correlated with decreased inflammation. Conversely, anti-TNF therapy increased IL-10 and IL-27 within the CNS without altering Foxp3+ Treg frequencies. Overall, these data demonstrate that TNF is an important mediator of the disability, inflammation, and pathology during progressive EAE. Our results demonstrate that TNF is a potential candidate to treat progressive forms of MS.
CD4+CD25+Foxp3+ cells from JNK-/- mice prolong pancreatic allograft survival in type 1 diabetic mice (TRAN2P.970)
Authors: Tripathi Deepak , Venkatasubramanian Sambasivan , Cheekatla Satyanarayana , Paidipally Padmaja , Welch Elwyn , Tvinnereim Amy , Vankayalapati Ramakrishna
Published: January 2023

In the current study we asked whether modified CD4+CD25+FoxP3+ regulatory T-cells (T-regs) can induce long term allograft tolerance. Pancreatic islets from BALB/c mice survived in a type 1 diabetic C57BL/6 mice (recipient mice) liver up to 18-21 days (n=5). Preloading islets with T-regs from WT (wild type) C57BL/6 mice, enhanced survival up to 37 days (N=5 and p = 0.003). Further preloading islets with T-regs from JNK1-/- mice (C57BL/6 background) enhanced survival up to 107 days (N=5 and p = 0.002). In contrast preloading islets with T-regs from FAS-/- (C57BL/6 background) did not enhance islet survival beyond 45 days. T-cell receptor stimulation of T-regs from JNK1-/- mice produced significant high amount of IL-10 (14.67 ± 2.784 vs 3.803 ± 0.8325, p = 0.009) and TGF-β (27.93 ± 7.563 vs 9.920 ± 1.212, p = 0.05) compared to T-regs from WT mice. Preloading islets with T-regs from JNK1-/- mice reduced IL-1β (30.91 ± 2.41 vs 16.13 ± 2.76; p = 0.003), TNF-α (3.802 ± 0.69 vs 1.888 ± 0.26; p = 0.03) IL-17 (24.82 ± 4.07 vs 4.708 ± 1.57; p = 0.0017). Studies are underway to determine the JNK1-/-T-regs mediated mechanisms involved in prolongation of allograft survival.
Deficiency of indoleamine 2,3-dioxygenase in donor bone marrow cells leads to exacerbation of graft-versus-host disease through enhanced generation of neutrophils and effector T cells (TRAN2P.965)
Authors: Ju Ji Min , Choi Eun
Published: January 2023

Indoleamine 2,3-dioxygenase-1 (IDO-1) is a tryptophan metabolic enzyme expressed by various types of cells including dendritic cells, myeloid-derived suppressor cells, and intestinal epithelial cells. The enzyme is also well known for immune suppressive function, while the underlying mechanism is still obscure. To understand cellular mechanism underlying the suppressive role of IDO-1, especially expressed by donor-derived myeloid cells in generation of graft-versus-host disease (GVHD), we transplanted T cell-depleted (TCD) BM cells from Ido-1-/- mice together with wild type (WT) T cells on C57BL/6 (B6) background into MHC-matched allogeneic BALB.B mice. Compared with BALB.B hosts that received WT TCD-BM and T cells, those of Ido-1-/- TCD-BM and WT T cells showed exacerbated GVHD. T cell frequencies in blood and target organs, and their productions of effector cytokines (IFN-γ- and IL-17) were enhanced in the recipients of Ido-1-/- TCD-BM, compared with those of WT TCD-BM. Generation of CD11b+Gr-1+ cells, especially of Ly6GhiLy6lo cells, was also enhanced in the formers. The CD11b+Gr-1+ cells originated from Ido-1-/- TCD-BM exerted lower suppressive activity over proliferation and cytokine production of co-incubated allo-reactive T cells. Proportions of CD244- neutrophils within the Ly6GhiLy6lo population of Ido-1-/- TCD-BM origin were significantly higher than that of WT TCD-BM. These results demonstrate implication of IDO-1 function in neutrophil generation.
Liver NK1.1 cells and IL-22 promote pancreatic islets allograft survival in type 1 diabetic mice (TRAN2P.969)
Authors: Tripathi Deepak , Venkatasubramanian Sambasivan , Cheekatla Satyanarayana , Paidipally Padmaja , Welch Elwyn , Tvinnereim Amy , Vankayalapati Ramakrishna
Published: January 2023

We determined the role of liver NK1.1 cells in allograft survival. Pancreatic islets from BALB/c mice survived in a type 1 diabetic C57BL/6 mice liver up to 15-21 days (n=5, p=0.001). Anti NK1.1 but not isotype control antibody treatment of type 1 diabetic C57BL/6 mice before and after pancreatic islet transfer rejected graft within 3 days (n=5, p=0.001). Anti-NK1.1 antibody treatment enhanced IL-1β (45.13 ± 5.9 vs 94.25 ± 5.5; p = 0.0003), TNF-α (4.746 ± 2.4 vs 11.92 ± 1.8; p = 0.047) and IFN-γ (2.714 ± 0.45 vs 4.344 ± 0.4; p = 0.036) and inhibited IL-22 (24.82 ± 4.07 vs 11.01 ± 2.186; p = 0.01) gene expression by liver lymphocytes of recipient mice. NK1.1 cells are major source of IL-22 in islets recipient mice liver and anti-IL-22 antibody treatment of recipient mice reduced allograft survival to 6-9 days compared to 18-21 days in isotype control antibody treated mice. Recombinant IL-22 but not IFN-γ enhanced insulin production by pancreatic islets of BALB/c mice (102.0 ± 22.65 vs 33.41 ± 12.07; p = 0.03) and enhanced expression of genes Reg2, Reg3a and Reg3g involved in islet survival. Further studies indicated that immunization and challenge of type 1 diabetic C57BL/6 mice with TLR9 agonist can enhance IL-22 production by NK1.1 cells and prolongs pancreatic allograft survival from 15 days to 45 days. The sum of our studies suggests that IL-22 produced by liver NK1.1 cells enhance allograft survival and TLR9 agonist can be used to prolong pancreatic allograft survival.
Expression of indoleamine 2.3-dioxygenase (IDO) by tumors induces local and systemic immunosuppressive effects in a murine melanoma model (TUM10P.1029)
Authors: Holmgaard Rikke , Zamarin Dmitriy , Munn David , Merghoub Taha , Wolchok Jedd
Published: January 2023

IDO is found in the majority of human tumors and has been described as an important contributor to the development of an immunosuppressive tumor microenvironment that blocks the action of cytotoxic antitumor effector T cells. In order to delineate the mechanisms of IDO-induced immunosuppression in melanoma, we generated B16 melanoma cell lines overexpressing IDO (B16-IDO). When implanted into mice, B16-IDO tumors exhibited aggressive tumor growth, characterized by more rapid tumor progression and local invasion. Furthermore, constitutive expression of IDO by B16 cells conferred resistance to T-cell-targeting immunotherapies, such as CTLA-4 and PD-1 blocking antibodies. Interestingly, the immunosuppressive effect of IDO overexpression was evident not just within the tumor, but systemically as well. Analysis of the tumors in vivo, showed that this effect was accompanied by changes favoring a highly inhibitory tumor microenvironment characterized by significant reduction on local tryptophan concentrations and lack of effector T cells at the tumor site. Most strikingly, we observed that immunosuppressive cell populations such as regulatory T cells and myeloid-derived suppressor cells dominated the cellular infiltrates of B16-IDO tumors. All together our findings delineates IDO expression as an important mechanism driving the institution of an inhibitory tumor microenvironment and tumor progression, and provides a strong rationale for therapeutic targeting of this pathway.
IL-22 induces colon cancer proliferation through STAT3 and PRC2 signaling (TUM10P.1036)
Authors: Sun Danfeng , Lin Yanwei , Kryczek Ilona , Zou Weiping
Published: January 2023

Background: IL-22(interleukin-22) is a newly identified cytokine which has been recently highlighted owing to its biological significance in carcinogenesis. However, the role of IL-22 in colon cancer proliferation has remained unclear. Since both JAK-STAT signaling and Polycomb Repression complex (PRC) 2 play crucial roles in epithelial proliferation. We investigated the mechanism by which IL-22 induces colon cancer proliferation. Methods: Colon cancer cells proliferation was detected by FACS and H3 Thymidine Incorporation. Cell cycle related genes were tested by Real-Time PCR. Lentiviral vector encoding gene specific shRNAs (STAT3, EZH2, SUZ12) and scramble particles were used to transfect colon cancer cells. ChIP assays were performed to detect the binding of different proteins and DNA. Results: IL-22 significantly induces colon cancer cells proliferation through STAT3 signaling. This increased proliferation involved the inhibition of p16 and p18 through aberrant promoter histone methylation H3K27me3. PRC2 catalyzes promoter H3K27me3 of p16 and p18, which can be up-regulated by IL-22. Furthermore, STAT3 can bind to PRC2 members (SUZ12 and EED), which also can be up-regulated by IL-22 stimulation. Conclusion: IL-22 can induce human colon cancer cells proliferation by activation of STAT3, which was phosphorylated and directed bind to the PRC2 complex, especially SUZ12 and EED, and consequently catalyzed the H3K27m3 of cell cycle check-point genes p16 and p18.
Induction of tissue-resident TFH-like cells in human cancer links monocyte activation to M2b macrophage polarization (TUM10P.1021)
Authors: Chen Min-Min , Kuang Dong-Ming
Published: January 2023

The existence, regulation, and functions of IL-21+ immune cells are poorly defined in human cancers. Here we identified a subset of protumorigenic IL-21+ TFH-like cells in human hepatocellular carcinoma. These cells were the major source of IL-21 in tumors and represented about 10% of the CD4+ T cell population at levels comparable to amounts of TFH cells present in lymph nodes. However, the TFH-like cells displayed a unique CXCR5−PD-1−BTLA−CD69high tissue-resident phenotype with substantial IFN-γ production, which differed from the phenotype of TFH cells. TLR4-elicited innate monocyte inflammation was essential for IL-21+ TFH-like cell induction in tumors, and activation of STAT1 and STAT3 was critical for TFH-like cell polarization in this process. Importantly, the TFH-like cells operated in IL-21-IFN-γ-dependent pathways to induce plasma cell differentiation and thereby create conditions for M2b macrophage polarization. Thus induction of TFH-like cells links innate inflammation to immune privilege in tumors.
Early stage human lung tumors give rise to a subpopulation of tumor-associated neutrophils with the composite characteristics of neutrophils and antigen-presenting cells (TUM10P.1042)
Authors: Eruslanov Evgeniy , Bhojnagarwala Pratik , Quatromoni Jon , Moon Edmund , Stephen Tom , Akimova Tatiana , Hancock Wayne , Conejo-Garcia Jose , Feldman Michael , Albelda Steven , Singhal Sunil
Published: January 2023

Tumor-associated neutrophils (TANs) represent a significant portion of inflammatory cells in the lung tumor; however, the role of TANs in cancer progression remains largely unexplored in humans. Given the key role of cytotoxic T lymphocytes in mediating anti-tumor immunity, the goal of this study was to identify subsets of TANs that might have specific roles in regulation of T cell responses in patients with lung cancer. Our analysis of 37 early-stage human lung tumors revealed two major sub-populations of TANs. One subset of “canonical” TANs expressed classic neutrophil markers. A second subset of “hybrid” TANs displayed a combination of neutrophil (Arg1+MPO+CD66b+CD15+) and antigen-presenting cell (CD14+HLA-DR+CCR7+CD86+) markers. These hybrid TANs comprised 0.5-25% of all TANs. Using human bone marrow (BM) cells, we identified conditions in which the immature granulocytes could be differentiated into cells that highly resemble hybrid TANs. Functionally, the BM-derived hybrid subset and hybrid TAN were able to: 1) stimulate antigen non-specific T cell proliferation, 2) induce the proliferation of allogeneic T cells, 3) trigger antigen-specific memory T cell responses, and 4) cross-present the NY-ESO peptide to TCR-transfected CD8 cells. Together, our results reveal a specialized subpopulation of TAN capable of triggering and supporting T cell responses. This property of hybrid neutrophils may provide new opportunities to boost the efficacy of current immunotherapy.
Defective TRAF3 modulates alternative NF-kB signaling and cytokine expression to promote cancer cell survival in HPV positive head and neck cancer (TUM10P.1049)
Authors: Zhang Jialing , Chen Hui , Yang Xinping , Guven Emine , Nussinov Ruth , Chen Zhong , VanWaes Carter
Published: January 2023

Persistent infection of high-risk human papillomaviruses (HPV) is a known pathogen associated with head and neck squamous cell carcinoma (HNSCC), especially in tonsil, oropharynx and oral cavity. The incidence of HPV+ HNSCC has been increased recently in the Unites States, while the molecular mechanism of virus promoting HNSCC has not been fully understood. Recently, The Cancer Genome Atlas (TCGA) project revealed unique homozygous and heterozygous deletions and mutations of TNF receptor-associated factor 3 (TRAF3) in HPV associated HNSCC tumors. Low expression of TRAF3 was observed in HPV positive HNSCC samples and cell lines with TRAF3 deletions. These tumors had elevated expression of NF-kB subunits involved in alternative pathway, such as NF-kB2 and RelB, often associated with increased NIK, cIAP1 and cIAP2 expression. Overexpression of TRAF3 in HPV+ cell lines significantly suppressed alternative NF-kB subunit NF-kB2/RELB nuclear localization, while increasing expression of IFNα/β and IL10 implicated in anti-viral immunity. Further, overexpression of TRAF3 inhibited cell growth, colony formation, and migration, and sensitized cells to TNF-a and cisplatin induced cell death. Our data revealed that defective TRAF3 activated alternative NF-kB signaling and suppressed anti-viral cytokine expression, which promoted cancer cell survival and drug resistance, especially in HPV+ HNSCC.
Inflammatory chemokine CCL3 Leads to enhanced antitumor development in the draining lymph node (TUM10P.1053)
Authors: Allen Frederick , Bobanga Iuliana , Barkauskas Deborah , Myers Jay , Huang Alex
Published: January 2023

CCL3 has been shown to be important in orchestrating cellular contacts in vaccinated lymph nodes. Moreover, dendritic cells can mediate both tumor tolerance and anti-tumor immunity in some tumor models. We hypothesize that incorporating CCL3 into tumor vaccines can enhance anti-tumor immunity. We inoculated naïve recipient mice with either WT or CCL3-secreting colon tumors. CCL3-secreting tumors showed enhanced antitumor immunity and suppressed growth kinetics in immunocompetent mice versus WT-tumors. Relative to non-injected controls or WT-injected tumors, 3 days after injections CCL3-secreting tumors revealed an increase of CD4+, CD8+, and CD19+ lymphocyte in the draining lymph node. Concurrently we conducted in vitro BMDCs experiments to assess the effects the tumor microenvironment (+/- CCL3) has on the capacity of DCs to upregulate T cell activation and adhesion molecules. BMDCs differentiated in the presence of CCL3 showed a greater capacity to upregulation CD86, ICAM1, CD11b, and CCR7 before and after LPS stimulation. Moreover, BMDCs cultured in the presence of WT and CCL3-secreting tumor supernatants revealed an increase in CD86, MHCII, and CCR7 before and after LPS stimulation. Together these data reveal that CCL3 in the tumor microenvironments leads to a reduction in tumor growth and early lymphocyte accumulation in the DLN, which may be a result of an enhanced capacity for DCs to home to the DLN and activate antitumor T cells.
Attenuated rabies virus promotes CNS immune infiltration and tumor necrosis in a mouse model of glioma (TUM10P.1059)
Authors: Bongiorno Emily , Hooper Douglas
Published: January 2023

Malignant glioma is resistant to conventional treatment regimens, and recent immunotherapeutic strategies have had limited success, presumably due to lack of immune cell infiltration and function in the brain. Attenuated rabies virus (RABV) is unique in its ability to allow the therapeutic infiltration of immune cells into the CNS, hence we speculated that infection might have a beneficial effect in the GL261 mouse model of glioma. We found that infection of mice with attenuated RABV after the establishment of a GL261 CNS tumor significantly prolongs their survival. Improved survival is associated with the appearance of tumor necrosis despite the inability of the virus to replicate in the tumor. Infected tumor bearing animals also have higher levels of GL261-specific IgG2A antibody, indicating that the Th1 arm of the response has been promoted. Increased B, T and NK cell infiltration as well as elevated IFNγ and ICAM expression is also observed in CNS tissues of infected mice. In addition tumor-associated cells bearing PD-L1, FasL, CD133, and M2 markers are strikingly less abundant in the infected mice compared to uninfected controls. Our results suggest that the tumor necrosis caused by infection with RABV is the result of altered cell activity within the tumor rather than a direct effect of the virus. Increased effector presence in the CNS combined with fewer regulatory and evasive tumor-associated cells contribute to better tumor killing in RABV-infected mice.