bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterizing the T-cell survival and differentiation during priming with downregulated γ-c cytokine/Jak3 signals (50.34)
Authors: Khattar Mithun , Wang Guohua , Miyahara Yoshihiro , Guo Zhiyong , Chen Wenhao , Stepkowski Stanislaw
Published: January 2023

T-cells play a central role in cell mediated immune responses to foreign antigens. Optimal T-cell activation and expansion requires antigen recognition via cognate T-cell receptors (TCRs) along with co-stimulatory and cytokine signals. The cytokine signals that lead to T-cell activation and proliferation, involve binding of common gamma-chain cytokines to their cognate receptors, that in turn activates Janus tyrosine kinase (Jak) 3, inducing cytokine gene transcription via the Jak/Stat (Signal Transducers and Activators of Transcription) pathway. However, it is currently unknown as to whether these paradigms of signaling during T-cell priming are universal to all T-cell subsets and lineages or unique to each. In the current study, we have examined the role of γ-c cytokine/Jak3 axis of signaling in survival and differentiation of different T-cell subsets during early phases of priming. We show highly reduced survival of CD8+ T-cells under cytokine/Jak3 inhibiting conditions during activation, whereas CD4+ T-cells were fairly more resistant. Loss of Jak3 signaling also inhibited the Treg differentiation while promoting pro-inflammatory Th17 cells thus, suggesting discrete and specific roles of this signaling pathway in different T-cell subsets and lineages. These findings contribute to our understanding of T-cell activation in different T-cell lineages as well as their differentiation into subsets, and are important for designing therapeutic approaches of the immune responses.
MEK Kinase 1 signaling regulates Natural Killer T cells (50.38)
Authors: Gallagher Ewen
Published: January 2023

MEK Kinase 1 (MEKK1) is of critical importance in controlling the thymus-dependent immune response by regulating Mitogen-Activated Protein Kinase (MAPK) activation from many receptors. Natural Killer T (NKT) cells are a distinct subset of lymphocytes that are characterized by the cell surface expression of a single invariant T-cell receptor (TCR) and Natural Killer (NK) cell markers. The activation of NKT cells is initiated by the engagement of the NKT receptor with glycolipid antigens presented by the molecule CD1d. Careful examination of Mekk1 kinase-deficient mice (Mekk1ΔKD) reveals multiple defects in NKT cells. Mekk1ΔKD NKT cells have deficient: MAPK signal transduction, proliferative expansion, apoptosis, and aberrant cytokine production. Mekk1ΔKD mice display elevated liver damage in a ConA-induced acute hepatitis liver injury model. My results demonstrate for the first time that MEKK1 is a critical MAPK Kinase kinase (MAP3K) signalling component for the regulation of NKT cells.
Control of T cell activation by Drak2 depends on protein kinase D and the interplay between calcium and reactive oxygen species (50.6)
Authors: Newton Ryan , Walsh Craig
Published: January 2023

Drak2 plays a negative regulatory role in acute T cell activation with direct consequences on proliferative capacity. The absence of Drak2 in T cells provokes responsiveness to sub-optimal stimuli, heightens calcium mobilization across a broad range of antigen receptor cross-linking, and increases production of Il-2, yet key survival factors are diminished within the context of Drak2 deficiency, ultimately limiting effector function. Increased co-stimulation, or transgenic expression of Bcl-xL, can rescue this proliferative defect. Thus, Drak2 provides a link between negative regulation of T cell activation and the requirement for adequate co-stimulation. The pathway through which this serine/threonine kinase functions has remained elusive. This work shows that Drak2 activity is dependent on protein kinase D (PKD), in a calcium-dependent manner, and that a PKD/Drak2 signaling axis serves to limit the influx of extracellular calcium following T cell activation. Furthermore, reactive oxygen species are required for Drak2 activity and are shown to play a key role in the initial phase of this negative feedback loop. Taken together, PKD and Drak2 are shown to limit the activation state of T cells through tuning those signals which initially converge to ultimately drive effector function.
PTPN4 and PTPN3 regulation of ITAM containing proteins (50.9)
Authors: Dougall David , van Oers Nicolai
Published: January 2023

The T cell receptor (TCR) uses a highly conserved ITAM signaling pathway to transduce intracellular signals following appropriate ligand binding. Activations of the ITAM signaling pathway is tightly controlled by diverse families of protein tyrosine kinases and protein tyrosine phosphatases. In earlier studies, we reported that two PTPases (PTPN3 and PTPN4) could directly dephosphorylate the tyrosine residues in the CD3 z ITAMs. However, T cells from both PTPN3 and PTPN4-deficient mice exhibit normal CD3 regulated ITAM signaling pathways. Since PTPN3 and PTPN4 have FERM domains that localize these enzymes to the cytoskeleton, it is possible that these PTPases target distinct proteins. Interestingly, moesin, ezrin, and dectin-1 are three cytoskeletal associated proteins that use ITAM sequences to transduce intracellular signals. To examine whether these distinct ITAM-containing proteins were dephosphorylated by PTPN3 or PTPN4, a combination of transient transfection assays and cell signaling studies in PTPase-deficient dendritic cells were performed. These experiments will reveal whether this PTPase-family dephosphorylates distinct ITAM-containing proteins.
Faithful epigenetic and transcriptional regulation of a transgenic human Th2 cytokine gene locus in the murine nuclear environment (51.1)
Authors: Pivniouk Vadim , Bailey Tom , Pivniouk Oksana , Kiesler Patricia , Wlasiuk Gabriela , Rosenbaum David , Kim Kenneth , Strempel Jannine , Daines Michael , Vercelli Donata
Published: January 2023

Dysregulated production of the Th2 cytokines IL4 and IL13 is a hallmark of allergy and asthma. Th2 cytokine gene locus regulation has been extensively studied in mice, much less in humans. We have generated mice carrying a 160 kb BAC transgene (BAC5) that encompasses human RAD50, IL13 and IL4 and includes all the elements known to be required for faithful regulation of mouse Th2 cytokine locus. BAC5 transgenic mice appeared normal, were born at the expected Mendelian ratio, and carried 1-15 intact copies of BAC5. In vitro polarized mouse transgenic CD4 Th2, but not Th1 cells expressed human IL13 and IL4, demonstrating tissue-specific regulation of human Th2 cytokines. Murine CD4 T cells expressed physiologic levels of human IL13 and IL4, particularly in a house dust mite immunization model. Human IL13 expression was transgene copy number-dependent, suggesting BAC5 includes the human Th2 locus control region. Notably, DNA methylation patterns at multiple DNase I hypersensitive sites in the Th2 locus of human CD4 T cells were highly comparable if not identical in transgenic CD4 T cells of equivalent differentiation stages. Our results support the notion that in homologous tissues, genetic sequence is largely responsible for directing transcriptional programs and chromatin modifications even in a heterologous nuclear environment. Moreover, our findings suggest BAC5 transgenic mice are a powerful tool to study human Th2 cytokine gene regulation and Th2-dependent events in vivo.
Characterization of a novel stimulation-dependent secondary promoter in the lymphotoxin alpha gene (51.12)
Authors: Posch Phillip , Yokley Brian
Published: January 2023

The lymphotoxin alpha (LTA) downstream region (DSR, +1 to +459) between the transcription and translation start sites contains polymorphisms that impact expression, yet the importance of the DSR to transcriptional regulation has not been examined. Similar regions in other genes have been shown to contain numerous regulatory elements and secondary promoter sites. We found that the LTA DSR is required for full activity of the LTA promoter in T and B cells as deletion of this region leads to a ≥55% reduction in luciferase expression under basal and stimulated conditions. Further, expression levels driven by the DSR alone were found to be comparable to that of the LTA regulatory region (-915 to +459) in basal and stimulated B and T cells. We hypothesized that the DSR contained a secondary promoter site that might consist of a putative Sp1 and/or TFII-I [initiator (Inr)-like element] binding sites located near the 3' end. Mutation of these sites resulted in significant reductions of DSR-mediated expression. ChIP assays showed that Sp1 and TFII-I, as well as RNA pol II, bind to this region of LTA in vivo. 5'RLM-RACE assays revealed LTA transcripts initiating from the Inr-like element when T cells were stimulated with human recombinant (hr) LT-α, hrTGF-β1 and hrKGF, but not when stimulated with PMA/ionomycin or left unstimulated. All transcripts were novel utilizing five novel start sites. These data show a complex stimulation-dependent pattern of LTA transcriptional regulation.
Alternatively spliced IL-4 protein is naturally secreted by T cells (51.2)
Authors: Bocharov Alexander , Lockatell Virginia , Lavania Sachin , Todd Nevins , Luzina Irina , Atamas Sergei
Published: January 2023

We have previously described an alternatively spliced isoform of IL-4 mRNA that omits exon 2 (IL-4δ2). We and others reported that the total levels of combined IL-4 + IL-4δ2 mRNAs and IL-4 / IL-4δ2 mRNA ratios change in patients with scleroderma, asthma, and various infections. Natural IL-4δ2 protein was not previously studied because most commercial anti-IL-4 antibodies (Ab) do not react with IL-4δ2. We established mammalian expression systems (HEK293 stably transfected or infected with recombinant adenoviral constructs) producing naturally processed (folded and glycosylated) human IL-4 or IL-4δ2. By screening a library of commercial anti-IL-4 Abs, we identified a selective Ab reacting exclusively with IL-4, and an indiscriminate Ab reacting equally with IL-4 and IL-4δ2. PBMC and T lymphocyte (> 95% purity) cultures from 3 donors with allergic asthma and 3 controls were stimulated with PMA/ionomycin for 24 hrs. The supernates were immunodepleted of wild-type IL-4 using four cycles of depletion with the selective antibody. These immunodepleted supernates were negative in ELISAs with the selective anti-IL-4 Ab. However, the IL-4-depleted supernates from asthmatics, but not controls, were positive in ELISAs with the indiscriminate antibody that now detected only IL-4δ2. The levels of IL-4δ2 were 5 to 10 fold lower than the levels of IL-4 in the initial undepleted supernates. Thus, IL-4δ2 is naturally produced not only in the mRNA form, but also as a secreted protein.
Nourexin-4 A Novel Anti-inflammatory Therapy for Influenza Flu (52.1)
Authors: Elgebaly Salwa , Perez Daniel , Sullivan Kathleen , Whitaker Craig , Caspe Stephanie , Yi Qiao , Kreutzer Donald
Published: January 2023

Relatively little is known about the inflammatory mediators and mechanisms that drive the progression of influenza flu infection to cytokine storm, lung dysfunction, organ failure, and ultimately death. Vaccines and antiviral medications cannot control the excessive host inflammatory response. We demonstrated the rapid release of a potent inflammatory mediator, recently named Nourin, by local mammalian tissues in response to injury and infection. Nourin is a formyl peptide that acts through the formyl peptide receptor (FPR) on phagocytic leukocytes. As an “initial signal” in the “innate immunity”, Nourin stimulates leukocyte chemotaxis, induce acute and chronic inflammation, and stimulates the release of cytokine storm mediators from monocytes and neutrophils. Nourin detected in plasma samples from patients with severe influenza infection was much higher compared to moderate influenza. We then tested the Nourin antagonist Nourexin-4, as specific competitive antagonist of formyl peptides on phagocytic leukocytes FPR. Nourexin-4 completely blocked neutrophil chemotaxis induced by the standard formyl peptide f-MLF and the host-derived Nourin released by (1) cultured epithelial cells infected with the H1N1 influenza virus (PR8) (6-24 hours), (2) Nourin detected in the serum of mouse model of H1N1 influenza (6 hrs), and (3) Nourin detected in severe and moderate influenza patients plasma samples. Nourexin-4 can be used to control virus-induced inflammation and protect patients.
Route of immunization influences the induction of humoral, cellular and protective immunity by live attenuated measles vaccine in rhesus macaques (52.11)
Authors: Lin Wen-Hsuan , Pan Chien-Hsiung , Adams Robert , Ford Brandi , Laube Beth , Mikszta John , Sullivan Vince , Griffin Diane
Published: January 2023

Measles is a leading cause of vaccine-preventable mortality worldwide. To facilitate vaccine distribution, aerosol immunization has been proposed, but the immune response induced, and protection afforded, by live attenuated measles virus (MV) vaccine (LAV) given by the respiratory route have not been systematically studied. Rhesus macaques were immunized with liquid or powder LAV through a nebulizer, an endotracheal tube, or parenterally by intramuscular injection. The method of immunization significantly influenced the induction of the humoral and cellular immune responses to LAV, but the effect on the cellular immune response was not correlated with the effect on the humoral response. For instance, nebulizer immunization induced good T cell, but poor antibody responses. Intratracheal challenge with wild type MV showed that respiratory immunization did not fully prevent infection. However, animals primed with nebulizer LAV showed accelerated viral clearance from both blood and respiratory tract that coincided with faster and higher levels of MV-specific recall CD4+/CD8+ T-cell responses compared to controls. Therefore, the route of immunization influences the induction of humoral, cellular and protective immune responses to measles vaccine. Protection correlates with neutralizing antibody and not with T-cell responses. Although, memory T cells alone are not sufficient for preventing MV infection, a robust recall T cell response does accelerate viral clearance.
Construction of multimerized scFv that neutralize Bovine Herpes Virus-1 (52.12)
Authors: Pasman Yfke , Nagy Eva , Kaushik Azad
Published: January 2023

Our laboratory earlier constructed functional antibody fragments, scFv (single chain fragment variable), with an 18 amino acid linker, against bovine herpes virus-1 (BoHV-1) (Koti et al. 2010. Molec. Immunol. in press). With an objective to enhance scFv avidity, the scFv in Vl-linker-VH configuration was constructed with a short flexible amino acid linker. An overlap PCR product comprising Vl-linker-VH was cloned into pPICZα vector and expressed in Pichia pastoris, under the influence of inducible AOX1 promoter. Affinity purified scFvs were analyzed by Western blot and mass spectrometry that confirmed the expected molecular weight of the scFv. Further, the multimerized scFvs, both purified and unpurified, neutralized BoHV-1 infection of MDBK cells in vitro. Taken together with previous studies, the scFvs with varying linker size are capable of effectively neutralizing BoHV-1. The scFv against BoHV-1 will provide protection at respriratory mucosal surfaces and prevent infection. In conclusion, scFvs both in multimerized and non-multimerized form provide a useful diagnostic and therapeutic agent against an important cattle pathogen. (Supported by NSERC Canada)
Totally synthetic nanoparticle vaccines produced via layer-by-layer assembly of polypeptide films induce protective T-cell responses (52.14)
Authors: Powell Thomas , Palath Naveen , DeRome Mary , Tang Jie , Jacobs Andrea , Boyd James
Published: January 2023

Nanoparticle vaccines were synthesized using layer-by-layer (LbL) deposition of oppositely-charged polypeptides to build up an artificial film on CaCO3 nanocores. Designed peptides (DP) containing antigenic epitopes of interest were incorporated into the layers of the film, yielding nanoparticles that delivered the DP payload to dendritic cells via phagocytosis. LbL nanoparticles induced DC maturation without triggering secretion of inflammatory cytokines including TNFα and IL-6. LbL nanoparticle delivery of DP to DC resulted in potent cross-presentation to CD8+ T-cells and more efficient presentation to CD4+ T-cells compared to presentation of soluble peptide. Mice immunized with a single dose of LbL nanoparticles without adjuvant mounted vigorous T-cell responses detectable by IFNγ and IL-4 ELISPOT and in vivo CTL assay. Mice immunized with nanoparticles containing ovalbumin-derived DP were protected from challenge with Listeria monocytogenes ectopically expressing ovalbumin. Similar to the in vitro DC maturation results, immunization with LbL nanoparticles did not induce inflammatory symptoms locally (injection site reaction) or systemically (serum cytokine spike). The potency and efficacy of polypeptide LbL nanoparticles administered in aqueous suspension without adjuvant or other formulation additive, and the absence of overt inflammatory responses, suggest that this strategy may be useful in producing novel vaccines against multiple diseases.
Mechanisms of post-exposure protection mediated by virus-like particles against Ebola virus infection (52.18)
Authors: Bradfute Steven , Tailor Prafullakumar , Anthony Scott , Ozato Keiko , Bavari Sina
Published: January 2023

Ebola virus causes hemorrhagic fever that is characterized by high viral titers, coagulopathy, and high lethality rates. Our lab has developed a virus-like particle (VLP) vaccine that is effective in mouse, guinea pig, and non-human primate models when given as a pre-exposure vaccine. In this work, we show that VLPs protect Ebola-infected mice when given 24 hours post-infection. We used this platform to analyze mechanisms of immunity for post-exposure protection against Ebola virus. Analysis of serum revealed a decrease in many cytokine levels in mice given VLPs post-exposure compared to infected, untreated mice. VLP-mediated post-exposure protection required B cells, perforin, STAT-1 signaling, and either CD4+ or CD8+ T cells. However, NK cells, IFN-gamma, and TNF-alpha were not required for this protection. Interestingly, transient depletion of macrophages or dendritic cells abrogated VLP-mediated post-exposure protection. Additional studies are underway to further characterize the mechanisms of immunity in this setting.
Preclinical evaluation of a 15-valent pneumococcal polysaccharide-protein conjugate vaccine in infant rhesus monkeys (52.20)
Authors: Skinner Julie , Indrawati Lani , Winters Michael , MacNair John , Manger Walter , Pujar Hari , Blue Jeffrey , Antonello Joseph , Heinrichs Jon , Caulfield Michael
Published: January 2023

The incidence of invasive pneumococcal disease (IPD) caused by the 91 serotypes of Streptococcus pneumoniae (PN) varies geographically and temporally as a result of changing epidemiology and vaccination. Prevnar® (Pfizer), the first licensed pneumococcal conjugate vaccine (PCV), comprises polysaccharides (PS) from 7 serotypes conjugated to the mutant diphtheria toxin carrier protein, CRM197. In the United States, this vaccine has been highly efficacious in reducing the incidence of IPD caused by vaccine serotypes, however, the incidence of non-vaccine serotypes (e.g., 19A, 22F, and 33F) has increased resulting in the need for vaccines with higher valencies. Indeed, 10- and 13-valent PCVs have recently been licensed. For expanded coverage, we have developed a 15-valent PCV containing PS from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to CRM197 and formulated on aluminum phosphate adjuvant. The vaccine was tested in infant rhesus monkeys since they, like human infants, do not respond to unconjugated PN PS. Infant (2-3 month old) rhesus monkeys were vaccinated 3 times with PCV-15 or Prevnar® at 2 month intervals, and serotype-specific IgG antibodies were measured using a multiarray electrochemiluminescence (ECL) assay. The results indicate that antibody responses to PCV-15 and Prevnar® were comparable for the 7 common serotypes and that post-vaccination responses to PCV-15 were > 10-fold higher than baseline for the 8 added serotypes.
Alphavirus particle filovirus vaccine activation of Dendritic cells (52.7)
Authors: Pisarcik Sarah , Herbert Andrew , Olinger Gene
Published: January 2023

Filoviruses, Marburg (MARV) and Ebola (EBOV), are among the deadliest etiological agents causing hemorrhagic fevers. Although little is known about the pathogenesis or what immunological responses are required to protect against filoviruses, a promising recombinant RNA replicon vaccine has been developed consisting of an attenuated alphavirus, Venezuelan equine encephalitis virus (VEE), to express MARV and EBOV viral genes such as the virus glycoprotein (GP). While numerous in vivo studies have demonstrated the protective efficacy of filovirus GP expressing VEE replicon particles (GP-VRP) in mice, guinea pigs, and non-human primates, when challenged with homologous virus, little has been done to understand the means by which these VRP filovirus vaccines initiate and establish immunity. In this study, we sought to dissect the protective mechanism of VRPs by evaluating the GP-VRPs effects on dendritic cells (DCs) by several characterization assays. Immunofluorescence assay (IFA), flow cytometry, and cytokine detection assays were used to assess maturation, co-stimulatory molecule expression, and cytokine expression profiles of GP-VRP-treated DCs. These findings will aid in deciphering early innate immune responses initiated by GP-VRP vaccination that are necessary for eliciting protective immunity against filovirus infection.
Placental extract suppresses mouse models of autoimmunity (83.4)
Authors: Landek-Salgado Melissa , Kimura Hiroaki , Rose Noel , Caturegli Patrizio
Published: January 2023

Pregnancy is known to modulate many autoimmune diseases. For example, autoimmune thyroiditis improves during pregnancy, and autoimmune hypophysitis presents during this period. The mechanisms underlying the effect of pregnancy on autoimmunity remain unknown. To determine whether placental proteins influence autoimmunity, we used a mouse model of experimental autoimmune hypophysitis (EAH). Here we report that co-immunization of mice with placental extract and pituitary extract, significantly decreased EAH incidence, severity, and antibody titers. Disease suppression was specific to the placenta; liver or muscle extracts were incapable of suppressing disease. Disease suppression, however, was not specific to EAH: co-immunization of placental extract with thyroglobulin, similarly decreased antibody titers. Immunization with pituitary extract during early or late pregnancy and administration of non-emulsified placental extracts were ineffective in suppressing EAH. Suppression of EAH was only observed when the placental extract was at the same location as the immunogen. Placental extracts were found to contain high levels of soluble TNF receptor one (sTNFR1). Furthermore, a placental extract deficient in TNFR1 was incapable of suppressing EAH, suggesting that sTNFR1 may be responsible for the placental-mediated suppression of EAH. These results suggest that a placental component, possibly sTNFR1, is capable of dampening the immune response to a co-injected immunogen.
Non-steroidal anti-inflammatory drugs blunt antibody production in human B lymphocytes from older volunteer (84.10)
Authors: Bancos Simona , Phipps Richard
Published: January 2023

Older individuals respond poorly to vaccination. B lymphocytes are responsible for the synthesis of antibodies that are generated in response to infection and vaccination. Our laboratory has shown that activated human B cells isolated from younger subjects (18-35 years old) express cyclooxygenase-2 (Cox-2) and that inhibition of Cox-2 by non-steroidal anti-inflammatory drugs (NSAIDs) blunts antibody production. However, NSAIDs are highly used by people over 60 years old. There is no information regarding Cox-2 expression in B cells in older human subjects (over 60 years old). We hypothesize that Cox-2 activity is reduced in B cells from older individuals. Young (18-35 years old) and older subjects (over 60 years old) were enrolled in the study. B cells were isolated from peripheral blood, stimulated in vitro with anti-Ig plus ODN CpG 2395 in the presence or absence of NSAIDs and Cox-2 expression, prostaglandin production, cytokines and antibody synthesis determined. Cell viability and proliferation were also assayed. Compared to younger subjects, B cells from older volunteers had reduced Cox-2 expression and also produced less antibody. Furthermore, in these individuals, antibody levels were further decreased by NSAIDs. Thus, NSAIDs and low intrinsic Cox-2 expression could be responsible for the poor antibody response to vaccination in the elderly.
Do Maternal Antibodies Inhibit Newborn Immunization? (84.15)
Authors: Akpinarli Akgul , Matzinger Polly
Published: January 2023

Investigations with measles vaccine suggested that persisting maternal antibodies might inhibit active immunization in the newborn. Because evolutionarily this makes no sense, it prompted us to revisit the relationship between maternal antibodies and neonatal immunity. We primed and boosted adult B6 mice against OVA and analyzed the level of OVA-specific antibodies in the serum of the progeny at various times after birth. The offspring of immunized mothers acquired high levels of antibodies, which lasted for the duration of lactation and then waned, disappearing at about 120 days for IgG1 and 50 days for IgG2a. Next, newborn mice were immunized after acquiring high titer of passively transferred antibody from their mother. We found that there was no apparent impairment of responsiveness as they made high titers of anti-OVA antibodies, and these did not wane, unlike those of their unimmunized littermates. Because the contribution of antibodies from the newborn could not be determined, we used a congenic marker system to discriminate maternal antibodies from those made by their progeny (Igha and Ighb). We found that newborn mice from both groups produced similar levels of Igha or Ighb antibodies against OVA. Therefore we conclude that newborn born immunization was not hindered by the presence of maternal antibodies.
Nocodazole induces apoptosis in human Jurkat T cells through G2/M arrest-dependent phosphorylation of Bcl-2 and Bim by p34cdc2 kinase and subsequent provoking mitochondrial death signaling pathway (85.16)
Authors: Kim Young Ho , Han Cho Rong , Jun Do Youn , Park Hae Sun , Woo Hyun Ju
Published: January 2023

Treatment of human Jurkat T cells with nocodazole, a microtubule-depolymerizing agent, provoked apoptosis along with G2/M-arrest, Bcl-2 phosphorylation at Thr-56 and Ser-70 residues, Bim phosphorylation, Δψm loss, and caspase cascade activation. Whereas these nocodazole-induced apoptotic events were abrogated by Bcl-2 overexpression, the G2/M-arrest and phosphorylation of Bcl-2 and Bim were sustained. Although Bcl-2 and Bim were mainly located in mitochondrial fraction regardless of their phosphorylation status, their physical interaction became reduced upon phosphorylation. FADD-positive Jurkat clone A3 and FADD-deficient Jurkat clone I2.1 exhibited similar susceptibilities to the apoptogenic activity of nocodazole. Further analysis using selective caspase inhibitors elucidated that the activation of caspase-9 and -3, leading to activation of caspase-7 and -8, was crucial for nocodazole-induced apoptosis. In the presence of the G1/S blocking agent (hydroxyurea), nocodazole failed to induce G2/M-arrest, phosphorylation of Bcl-2 and Bim, and mitochondria-dependent apoptotic events. Nocodazole-induced phosphorylation of Bcl-2 and Bim, Δψm loss, and apoptotic events were significantly reduced by pretreatment with p34cdc2 kinase inhibitor (roscovitine). Consequently, these results indicated that G2/M-arrest and resultant phosphorylation of Bcl-2 and Bim dictated by p34cdc2 kinase, contributed to mitochondria-dependent apoptosis in Jurkat T cells exposed to nocodazole.
Regulation of TRAIL Expression in T helper cells (85.17)
Authors: Zhang Jimin , Shi Yufang
Published: January 2023

TNF-related apoptosis-inducing ligand (TRAIL/Apo2 ligand), along with FasL and TNFα, is one of three members of the TNF cytokine family that induce apoptosis. TRAIL can bind to several different receptors including those containing a cytoplasmic death domain and others that lack such a domain, termed decoy receptors. Here we found that TRAIL was preferentially expressed in Th2 cells, and, though exogenous TRAIL did not cause death in these cells, it did induce apoptosis of Th1 cells. Since it differentially induced apoptosis in Th1 and Th2 cells, TRAIL was likely to modulate the balance between them in determining the type of immune response. However, the regulation mechanism of TRAIL expression in T lymphocytes is not fully understood. We showed that A1.1 T hybridoma cells constitutively expressed TRAIL at a low level that could be boosted by adding IFN-γ. When Th2 cells were treated with high dosage of IFN-γ upon restimulation, TRAIL expression was boosted as well, whereas when Th1 cells were treated with high dosage of IL-4 upon restimulation, TRAIL was surprisingly detected at a comparable level to that in Th2 cells without any cytokine treatment. Interestingly, although Th1 cells were the main producers of IFNγ, Th2 cells expressed IFNγ receptors. All these results suggest that IFN-γ secreted by Th1 cells and IL-4 secreted by Th2 cells may be potent inducers of TRAIL under certain circumstances and therefore can provide some insight into clinical applications.
Deciphering the function of the microRNA-181a in effector CD4+ T cells (85.19)
Authors: Deng Min , Arias Marco , Ramaswamy Madhu , Siegel Richard
Published: January 2023

One of the mechanisms of peripheral tolerance in CD4 T cells, mediated by the TNF superfamily member Fas/FasL, occurs through a process termed Restimulation Induced Cell Death. Previously, we showed that naïve and memory activated CD4 T cell subsets vary in their response to Fas apoptosis, with the effector memory subsets having the highest Fas sensitivity. Since this phenotype was maintained stably in activated cell cultures, genetic or epigenetic regulation is likely. To examine the role of miRNAs in regulating the phenotype of effector memory (Em) T cells, we performed a miRNA array via Solexa sequencing to profile miRNAs differentially expressed in CD4 T cell subsets. One of these, miR181a, was upregulated in naïve and Cm, but down modulated in memory and Em cells. This downregulation correlated with the upregulation of its target genes including DUSP6. Mir181a is required for positive selection of T cells in the thymus, however, its function in peripheral TCR signaling is unknown. To probe the functional consequences of differential miR181a expression in peripheral T cell subsets we utilized a siRNA knockdown and lentiviral overexpression approach. Thus far we have not observed overt differences in Fas sensitivity, however, we have seen skewing of cytokine responses in miR181a knockdown, including changes in IL-17 and IFN-g expression following TCR triggering. These results suggest a role for miR-181a in regulating certain aspects of the effector memory T cell phenotype.
Human pluripotent stem cells: a source of mast cells for the study of allergic and inflammatory diseases (86.16)
Authors: Kovarova Martina , Latour Anne , Chason Kelly , Tilley Stephen , Koller Beverly
Published: January 2023

Human mast cells are tissue resident cells with a principal role in allergic disorders. Cross-linking of the high-affinity receptor for IgE (FcϵRI) results in release of inflammatory mediators initiating the clinical symptoms of allergy and anaphylaxis. Much of our knowledge regarding the mechanisms of mast cell activation comes from studies of mouse bone marrow derived mast cells. However, clear differences have been identified between human and mouse mast cells. Studies of human mast cells are hampered by the limited sources available for their isolation, the resistance of these cells to genetic manipulation, and differences between cultures established from different individuals. To address this limitation, we developed a simple co-culture-free method for obtaining mast cells from human pluripotent stem cells (hPS) including human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells. hES-derived mast cells respond to antigen by releasing mast cell mediators. Moreover, the cells can be generated in numbers sufficient for studies of the pathways involved in their effector functions. Genetically modified mast cells expressing GFP and Neo resistance were obtained by modification of hES cells prior to differentiation. iPS cells can be used for derivation of mast cells with patient specific genetic make-up. This direct co-culture-free differentiation of hPS cells represents a new and unique model to analyze the function and development of human mast cells.
Regulation of RGS13 expression by the tumor suppressor p53 in mast cells (86.22)
Authors: Druey Kirk , Lu Yunbiao , Kuehn Hyesun , Gilfillan Alasdair , Xie Zhihui
Published: January 2023

Regulators of G-protein Signaling (RGS) proteins negatively regulate GPCRs by accelerating the GTPase activity of Gα. In recent years, non-GPCR-related functions have also been described for these proteins. We found previously that RGS13 was enriched in mast cells and inhibited anaphylaxis in mice by suppressing IgE-mediated mast cell degranulation. We therefore examined the transcriptional regulation of RGS13 in mast cells. We confirmed the transcriptional start site (TSS) by 5’ RACE in cDNA from the human mast cell line LAD2 and demonstrated promoter activity in a ~ 1 kb element upstream of exon 1 using luciferase reporter assays. We also identified 2 potential p53 response elements (RE) in the region 2 kb upstream of the TSS by bioinformatic analysis. Overexpression and shRNA-mediated knockdown of endogenous p53 in LAD2 cells revealed an inverse relationship between p53 levels and RGS13 expression. Endogenous p53 in these cells bound exclusively to oligonucleotides containing the p53 RE most upstream of the TSS. Cells expressing double-stranded oligonucleotides containing this site or a p53 consensus site had enhanced promoter activity. These studies indicate that p53 suppresses RGS13 transcription in mast cells. Thus, upregulation of p53 activity in mast cells exposed to stress could reduce the threshold for degranulation as a result of decreased RGS13 expression.
Effects of a novel mucosal tolerogen on critical T cell cytokine secretion and development of anti-FVIII inhibitor Ab in a mouse model of Hemophilia A (87.27)
Authors: Csencsits-Smith Keri , Nguyen Trinh , Donnachie Elizabeth , Borck Margarete , Escobar Miguel , Pascual David
Published: January 2023

The development of anti-FVIII (F8) antibodies (Abs) in Hemophilia A patients who receive therapeutic supplemental F8 results in significant morbidity. T cell help is required for anti-F8 inhibitor formation, however, the role of secreted cytokines is not yet fully understood. To ascertain the T cell cytokines important in the development of inhibitor Abs, F8-deficient mice were immunized 4x with 2μg recombinant human F8 at weekly intervals. To abate the production of inhibitor Ab, mice were intranasally immunized with 100μg of E. coli colony-forming antigen I (CFA/I), a novel tolerogen which reduces inflammatory immune responses. Plasma was assayed for the presence of secreted cytokines via multiplex and for functional anti-F8 Ab via Bethesda assay. Inhibitor Ab was detectable by day 14 post-primary immunization, and Th1 (IFNγ and IL-12), Th2 (IL-4), and Th17 (IL-17) cytokines were present in control and CFA/I treated plasma as early as 7 days post-primary immunization. There was no apparent correlation between Th1 vs Th2 responses and increased inhibitor titer. However, mice that were resistant to the effects of CFA/I treatment displayed increased anti-F8 Ab as well as IL-6 and IL-17 by day 21 post-primary immunization. In contrast, IL-13 secretion was associated with significantly decreased inhibitor antibody in CFA/I treated mice. Our results suggest that modification of Th cytokine response may amerliorate inhibitor Ab formation.
Adenosine A2A receptor activation inhibits T cell activity in a murine model of chronic granulomatous disease (87.3)
Authors: Lappas Courtney , Domeier Phillip , Chehata Veronica
Published: January 2023

Chronic granulomatous disease (CGD) is caused by defects in the NADPH oxidase system, and is characterized by an increased susceptibility to recurrent, severe bacterial and fungal infections. Early diagnosis and aggressive treatment can be effective at combating life threatening infections, but other significant complications of CGD, such as non-infectious hyperinflammatory manifestations, result in a high associated morbidity. Recent studies suggest that the hyperinflammation observed in CGD (gp91phox deficient) mice may be due in part to dysregulated T cell activity. We have broadly characterized the phenotypes of T lymphocytes collected from p91phox deficient mice and our data indicate that although the basal activation states of T cells collected from WT and CGD mice are similar, upon activation through the T cell receptor, CGD T cells produce higher levels of the pro-inflammatory cytokines IFN-γ and IL-2 and express elevated levels of the cell surface activation markers CD25, CD69 and CD40L as compared to WT T cells. This hyper-activation of CGD T cells is effectively modulated by treatment with the selective adenosine A2A receptor agonist CGS21680, which reduces the levels of IFN-γ and IL-2 by approximately 90% and down regulates CD25, CD69 and CD40L expression by approximately 50%. These data suggest that the adenosine A2A receptor is a potential pharmacological target for the treatment of the hyperinflammatory complications observed in CGD patients.
Identification of an XBP-1-Binding Endoplasmic Reticulum Stress-Responsive Enhancer of IFN-β Transcription in Macrophages (88.11)
Authors: Smith Judith , Liu Yi-Ping , Chen Hui , Qi Ling , Zeng Ling
Published: January 2023

Perturbation of the endoplasmic reticulum (ER) by various insults results in a conserved stress response called the “Unfolded Protein Response” (UPR). We have previously shown that macrophages undergoing a UPR respond to LPS with log-fold increased production of IFN-β a cytokine with diverse roles in innate and adaptive immunity. In this study, we found that pre-treatment of macrophages with the ER stress inducer thapsigargin resulted in augmented recruitment of IRF-3, CBP/p300, and ultimately transcriptional machinery, to the ifnb1 gene promoter during LPS stimulation. Although full synergistic IFN-β production required XBP-1, this UPR regulated transcription factor does not bind directly to the ifnb1 promoter. Analysis of the genomic DNA surrounding the ifnb1 gene revealed a conserved site 6.1kb downstream of ifnb1 that bound XBP-1 along with IRF-3, and CBP only during concomitant UPR and LPS stimulation. XBP-1 co-precipitates with p300, suggesting a mechanism of multi-molecular assembly. Results from luciferase reporter assays provide evidence that this +6kb region functions as an XBP-1-dependent enhancer of ifnb1 promoter activity. These findings provide a mechanistic link for diseases involving ER stress and type I interferon, including viral infection, ischemia-reperfusion injury, protein-misfolding and inflammatory diseases.
Distal regions of the human IFNG locus direct cell-type specific expression (88.12)
Authors: Collins Patrick , Henderson Melodie , Chang Shojing , Davis Georgia , McLoed Allyson , Mortlock Douglas , Aune Thomas
Published: January 2023

Genes, such as IFNG, which are expressed in multiple cell lineages of the immune system, may employ a common set of regulatory elements to direct transcription in multiple cell types or individual regulatory elements to direct expression in individual cell lineages. By employing a BAC transgenic system, we demonstrate that IFNG employs unique regulatory elements to achieve lineage specific transcriptional control. Specifically, a one 1-kb element 30 kb upstream of IFNG activates transcription in T cells and NKT cells but not NK cells, macrophages and dendritic cells. This distal regulatory element is a Runx3 binding site in Th1 cells, and is needed for RNA polymerase II recruitment to IFNG, but not for histone acetylation of the IFNG locus. These results support a model whereby IFNG utilizes distinct regulatory elements to achieve cell-type expression.
DNMT3a mediates lineage-specific, de novo DNA methylation at the ifng promoter and contributes to ifng gene silencing in Th2, Th17 and Treg cells (88.15)
Authors: Thomas Rajan , Gamper Christopher , Powell Jonathan , Wells Andrew
Published: January 2023

Naïve CD4+ T cells can differentiate into distinct subsets of effectors that express unique cytokine profiles upon activation. T helper 1 (Th1) cells robustly secrete IFNγ, a cytokine essential for clearance of intracellular pathogens and anti-tumor responses, while Th2, Th17, and regulatory T cells (Tregs) do not. The silencing of “off-pathway” cytokine genes during differentiation is incompletely understood, but may involve epigenetic mechanisms of gene repression. CpG dinucleotides in the ifng promoter, which are largely unmethylated in naïve CD4+ T cells and Th1 cells, are sites of substantial de novo methylation in non-IFNγ-producing lineages. To determine whether such de novo DNA methylation contributes to ifng gene silencing, we used mice with T cell-specific deletion of the de novo DNA methyltransferase, DNMT3a. While “on-pathway” cytokine expression polarized normally, DNMT3a-deficient CD4 T cells did not accumulate de novo ifng promoter methylation seen in wild-type cells during Th2, Th17 or Treg differentiation. Maintenance DNA methylation at the ifng intronic enhancer was not affected. Failed de novo methylation was associated with failed silencing, as DNMT3a-deficient Th2, Th17 and Treg cells produce significant levels of IFN-γ after secondary re-polarization toward the Th1 lineage. These results indicate that DNMT3a restricts the plasticity of CD4+ T helper cell fates via de novo DNA methylation of the ifng promoter in Th2, Th17 and Treg cells.
E2F1 Suppreses DC Maturation (88.22)
Authors: Xiao Weihua , Fang Fang , Wang Yan , Li Rui , Zhao Ying , Yang Guo , Jiang Ming , Sun Jie , Ma Yang , Ren Zijia , Tian Zhigang , Feng Wei , Yang De
Published: January 2023

E2F1 has been largely studied as a promoter of cell cycle and as regulator of apoptosis. Recently, E2F1 has been shown to regulate a wide range of genes in response to inflammatory stimulation of macrophages and to contribute to T cell activation in response to pathogens, implicating an extensive immunological role for E2F1. Dendritic cells (DCs) play critical roles as professional antigen-presenting cells in the development of immune responses, however, it is unclear whether E2F1 has any effect on DC phenotype and function. Here, we report that E2F1 acts as a suppressor of DC maturation. The level of E2F1 expression was transiently downregulated in the course of LPS-induced maturation of both human monocyte-derived dendritic cells and a mouse DC cell line, DC2.4. Knockdown of E2F1 by siRNA in DC2.4 cells resulted in both phenotypic and functional maturation, even without LPS treatment. Conversely, ectopic overexpression of E2F1 suppressed LPS-induced maturation of DC2.4 cells. Furthermore, knockdown of E2F1 caused the activation of several major signaling pathways known to be activated in the course of DC maturation, including Erk1/2, NF-κB and PI3K/Akt, suggesting that E2F1 may be involved in regulating multiple signaling pathways in DCs. Finally, the alternation by E2F1 on phenotypic maturation was confirmed with bone marrow-derived DCs from E2F1-KO mice. Overall, our data demonstrate for the first time that E2F1 is a critical regulator of dendritic cell maturation.
Role of Dnmt3a in cytokine gene regulation during Th1/Th2 differentiation (88.23)
Authors: Yu Qing , Kaplan Mark
Published: January 2023

Helper T cell polarization is accompanied by DNA methylation-mediated silencing of specific genes. Dnmt3a/3b, de novo DNA methyltransferases, have been shown to associate with cytokine gene promoters during Th1/Th2 differentiation. To elucidate the functional significance of Dnmt3a/3b-mediated DNA methylation in gene regulation of CD4 cell development, we generated conditional knockout mice by crossing Dnmt3aloxP/loxP and Dnmt3bloxP/loxP mice (a gift from Dr. En Li) with CD4-CRE mice. The conditional knockout mice exhibited normal lymphoid organ cellularity. Analysis of gene expression revealed that Dnmt3a-deficient Th1/Th2 cells secreted higher levels of effector cytokines in comparison with littermate controls, whereas absence of Dnmt3b did not show significant change, compared to controls. Using a DNA methylation-sensitive restriction method, we found that although absence of Dnmt3a did not alter global methylation levels of CD4 cells, Dnmt3a deficiency led to reduced levels of DNA methylation at specific gene loci, indicating that Dnmt3a participated in the establishment of local DNA methylation. Meanwhile, Chromatin IP data showed histone modification alterations in the absence of Dnmt3a. Taken together, the results indicated that Dnmt3a had an inhibitory effect on gene expression in Th1/Th2 development.
Arsenic enhances Langerhans cell migration to draining lymph nodes via CCL21 and IFN-γproduction in a model of epicutaneous protein sensitization (89.19)
Authors: Lee Chih-Hung , Hong Chien-Hui , Yu Chia-Li , Wang Li-Fang , Liao Wei-Ting , Huang Shau-Ku , Chen Gwo-Shin , Yu Hsin-Su
Published: January 2023

Arsenic induces multiple Bowen's disease (As-BD), or cutaneous carcinoma in situ, that runs an indolent course. Immune responses are important modifiers for the development of As-BD. We have previously reported that Langerhans cells (LCs) were reduced in As-BD, but how arsenic alters LC migration and polarizes Th responses remains unknown. Using an epicutaneous protein sensitization model, Balb/c mice were patch-sensitized with OVA and received feeding of 300 ppm arsenic or PBS for 200ul for 5 days. Defined as CD207+MHCII+ cells, the number of LCs was significantly decreased in patched epidermis, but increased in draining lymph nodes, with no changes in dermis and spleens 96 hours after OVA sensitization in arsenic-treated mice. There was a parallel increase of CCL21 in lymph nodes from arsenic-treated mice, and enhanced cellular proliferation and IFN-γ secretion in vitro were also noted in lymph node cells from arsenic-treated mice. Notably, in vivo administration of a neutralizing antibody against CCL21 abolished the increase of LCs in lymph nodes. These results suggest, therefore, that in this model, arsenic exposure enhanced LC migration to draining lymph nodes via CCL21 and interferon-γ production, and that chronic nature of As-BD might result from enhanced Th1 responses and LC migration.