bluuebunny/crossref_metadata_2025 huggingface dataset:
4-1BB Promotes the Survival of CD8+ T Lymphocytes by Increasing Expression of Bcl-xL and Bfl-1
Authors: Lee Hyeon-Woo , Park Su-Jung , Choi Beom K. , Kim Hyun Hwa , Nam Kyung-Ok , Kwon Byoung S.
Published: April 2014

4-1BB, a T cell costimulatory receptor, prolongs CD8+ T cell survival. In these studies, 4-1BB stimulation was shown to increase expression of the antiapoptotic genes bcl-xL and bfl-1 via 4-1BB-mediated NF-κB activation. This signaling pathway was specifically inhibited by PDTC and was different from the pathways that enhanced CD8+ T cell proliferation. The results suggest a role for the antiapoptotic activities of Bcl-xL and Bfl-1 proteins in 4-1BB-mediated CD8+ T cell survival in vivo.
CD28, TNF Receptor, and IL-12 Are Critical for CD4-Independent Cross-Priming of Therapeutic Antitumor CD8+ T Cells
Authors: Hu Hong-Ming , Winter Hauke , Ma Jun , Croft Michael , Urba Walter J. , Fox Bernard A.
Published: April 2014

Previously, we have shown that priming of therapeutic CD8+ T cells in tumor vaccine-draining lymph nodes of mice vaccinated with GM-CSF secreting B16BL6 melanoma cells occurs independent of CD4 T cell help. In this study, we examined the contribution of the major costimulatory molecules, CD40 ligand (CD40L), CD80, and CD86, in the priming of CD8+ T cells. Priming of therapeutic CD8+ T cells by a GM-CSF-transduced tumor vaccine did not require CD40 and CD40L interactions, as therapeutic T cells could be generated from mice injected with anti-CD40L Ab and from CD40L knockout mice. However, costimulation via either CD80 or CD86 was required, as therapeutic T cells could be generated from mice injected with either anti-CD80 or anti-CD86 Ab alone, but administration of both Abs completely inhibited the priming of therapeutic T cells. Blocking experiments also identified that priming of therapeutic T cells in MHC class II-deficient mice required TNFR and IL-12 signaling, but signaling through CD40, lymphotoxin-βR, or receptor activator of NF-κB was not essential. Thus, cross-priming of therapeutic CD8+ T cells by a tumor vaccine transduced with GM-CSF requires TNFR, IL-12, and CD28 signaling.
Positive Selection by the Pre-TCR Yields Mature CD8+ T Cells
Authors: Ito Yuriko , Arai Satoko , van Oers Nicolai S. C. , Aifantis Iannis , von Boehmer Harald , Miyazaki Toru
Published: April 2014

It has been of much interest whether there is functional redundancy between the constitutively signaling pre-Tα/TCRβ (pre-TCR) and ligated TCRαβ complexes, which independently operate the two distinct checkpoints during thymocyte development, i.e., the pre-TCR involved in β-selection at the CD4−CD8− double-negative stage and the TCRαβ being crucial for positive/negative selection at the CD4+CD8+ double-positive stage. We found that the pre-TCR expressed on double-positive cells in TCRα-deficient (TCRα−/−) mice produced a small number of mature CD8+ T cells. Surprisingly, when pre-Tα was overexpressed, resulting in augmentation of pre-TCR expression, there was a striking increase of the CD8+ T cells. In addition, even in the absence of up-regulation of pre-TCR expression, a similar increase of CD8+ T cells was also observed in TCRα−/− mice overexpressing Egr-1, which lowers the threshold of signal strength required for positive selection. In sharp contrast, the CD8+ T cells drastically decreased in the absence of pre-Tα on a TCRα−/− background. Thus, the pre-TCR appears to functionally promote positive selection of CD8+ T cells. The biased production of CD8+ T cells via the pre-TCR might also support the potential involvement of signal strength in CD4/CD8 lineage commitment.
The Role of Fibroblasts in Thymocyte-Positive Selection
Authors: Lilić Mirjana , Santori Fabio R. , Neilson Eric G. , Frey Alan B. , Vukmanović Stanislav
Published: April 2014

Mice with fibroblast-specific expression of TAP-1 were generated by expressing the TAP-1 transgene under the control of the fibroblast-specific protein (FSP) 1 promoter/enhancer on TAP-1-deficient background. MHC class I expression in primary fibroblast cultures isolated from the resulting strain mimicked that of wild-type counterparts. MHC class I was detected in both types of fibroblasts following treatment with IFN-αβ. Positive selection of CD4−CD8+ thymocytes was observed in neither adult nor fetal/neonatal thymus of transgenic mice. IFN-αβ-induced expression of MHC class I rescued positive selection of CD4−CD8+ T cells in fetal thymic organ cultures, but not in adult mice. Contrary to previous suggestions, our results indicate a limited role of fibroblasts in promoting positive selection. In addition, the results suggest that positive selection may occur by a different mechanism in fetal vs adult thymus.
Modulation of Gene Expression by the MHC Class II Transactivator
Authors: Nagarajan Uma M. , Bushey Alyssa , Boss Jeremy M.
Published: April 2014

The class II transactivator (CIITA) is a master regulator of MHC class II expression. CIITA also modulates the expression of MHC class I genes, suggesting that it may have a more global role in gene expression. To determine whether CIITA regulates genes other than the MHC class II and I family, DNA microarray analysis was used to compare the expression profiles of the CIITA expressing B cell line Raji and its CIITA-negative counterpart RJ2.2.5. The comparison identified a wide variety of genes whose expression was modulated by CIITA. Real time RT-PCR from Raji, RJ2.2.5, an RJ2.2.5 cell line complemented with CIITA, was performed to confirm the results and to further identify CIITA-regulated genes. CIITA-regulated genes were found to have diverse functions, which could impact Ag processing, signaling, and proliferation. Of note was the identification of a set of genes localized to chromosome 1p34-35. The global modulation of genes in a local region suggests that this region may share some regulatory control with the MHC.
Analysis of MHC Class II Antigen Processing by Quantitation of Peptides that Constitute Nested Sets
Authors: Lippolis John D. , White Forest M. , Marto Jarrod A. , Luckey Chance J. , Bullock Timothy N. J. , Shabanowitz Jeffrey , Hunt Donald F. , Engelhard Victor H.
Published: April 2014

Peptides associated with class II MHC molecules are of variable length because in contrast to peptides associated with class I MHC molecules, their amino and C termini are not constrained by the structure of the peptide interaction with the binding site. The proteolytic processing events that generate these peptides are still not well understood. To address this question, peptides extracted from HLA-DR*0401 were analyzed using two types of mass spectrometry instrumentation. This enabled identification of >700 candidate peptides in a single analysis and provided relative abundance information on 142 peptides contained in 11 nested sets of 3–36 members each. Peptides of 12 residues or less occurred only at low abundance, despite the fact that they were predicted to fully occupy the HLA-DR*0401 molecule in a single register. Conversely, the relative abundance of longer species suggested that proteolytic events occurring after MHC binding determine the final structure of most class II-associated peptides. Our data suggest that C-terminal residues of these peptides reflect the action of peptidases that cleave at preferred amino acids, while amino termini appear to be determined more by proximity to the class II MHC binding site. Thus, the analysis of abundance information for class II-associated peptides comprising nested sets has offered new insights into proteolytic processing of MHC class II-associated peptides.
The HLA Molecules DQA1*0501/B1*0201 and DQA1*0301/B1*0302 Share an Extensive Overlap in Peptide Binding Specificity
Authors: Sidney John , del Guercio Marie-France , Southwood Scott , Sette Alessandro
Published: April 2014

Assays to measure the binding capacity of peptides for HLA-DQA1*0501/B*0201 (DQ2.3) and DQA1*0301/B*0302 (DQ3.2) were developed using solubilized MHC molecules purified from EBV-transformed cell lines. These quantitative assays, based on the principle of the inhibition of binding of a high-affinity radiolabeled ligand, were validated by examining the binding capacity of known DQ-restricted epitopes or ligands. The availability of these assays allowed an investigation of patterns of cross-reactivity between different DQ molecules and with various common DR molecules. DQ2.3 and DQ3.2 were found to have significantly overlapping peptide binding repertoires. Specifically, of 13 peptides that bound either DQ2.3 or DQ3.2, nine (69.2%) bound both. The molecular basis of this high degree of cross-reactivity was further investigated with panels of single substitution analogs of the thyroid peroxidase 632–645Y epitope. It was found that DQ2.3 and DQ3.2 bind the same ligands by using similar anchor residues but different registers. These data suggest that in analogy to what was previously described for HLA-DR molecules, HLA-DQ supertypes characterized by largely overlapping binding repertoires can be defined. In light of the known linkage of both HLA-DQ2.3 and -DQ3.2 with insulin-dependent diabetes mellitus and celiac disease, these results might have important implications for understanding HLA class II autoimmune disease associations.
Activated Intrahepatic Antigen-Presenting Cells Inhibit Hepatitis B Virus Replication in the Liver of Transgenic Mice
Authors: Kimura Kiminori , Kakimi Kazuhiro , Wieland Stefan , Guidotti Luca G. , Chisari Francis V.
Published: April 2014

In this study we evaluated the ability of activated intrahepatic APCs to inhibit hepatitis B virus (HBV) replication in transgenic mice. Intrahepatic APCs were activated by administration of an anti-CD40 agonistic mAb (αCD40). We showed that a single i.v. injection of αCD40 was sufficient to inhibit HBV replication noncytopathically by a process associated with the recruitment of dendritic cells, macrophages, T cells, and NK cells into the liver and the induction of inflammatory cytokines. The antiviral effect depended on the production of IL-12 and TNF-α by activated APCs; however, it was mediated primarily by IFN-γ produced by NK cells, and possibly T cells, that were activated by IL-12. Collectively, these results suggest that activated APCs can directly produce antiviral cytokines (IL-12, TNF-α) and trigger the production of other cytokines (i.e., IFN-γ) by other cells (e.g., NK cells and T cells) that do not express CD40. These results provide insight into a hitherto unsuspected antiviral function of intrahepatic APCs, and they suggest that therapeutic activation of APCs may represent a new strategy for the treatment of chronic HBV infection.
Perforin-Mediated CTL Cytolysis Counteracts Direct Cell-Cell Spread of Listeria monocytogenes
Authors: San Mateo Lani R. , Chua Ming Ming , Weiss Susan R. , Shen Hao
Published: April 2014

The immune system has evolved various effector cells and functions to combat diverse infectious agents equipped with different virulence strategies. CD8 T cells play a critical role in protective immunity to Listeria monocytogenes (Lm), a bacterium that grows within the host cell cytosol and spreads directly into neighboring cells. The importance of CD8 T cells during Lm infection is currently attributed to the cytosolic niche of this organism, which allows it to evade many aspects of immune surveillance. CTL lysis of infected cells is believed to be an essential protective mechanism, presumably functioning to release intracellular bacteria, although its precise role remains to be fully defined. In this study, we examined the contribution of perforin-mediated CTL cytolysis to protective immunity against recombinant Lm capable of or defective in cell-cell spread. We found that CTL cytolysis is critical for protective immunity to Lm capable of cell-cell spread while protective immunity against spread-defective Lm is largely independent of CTL cytolysis. These results demonstrate that an important function of CTL cytolysis is to counter the microbial virulence strategy of direct cell-cell spread. We propose a model that advances the current view of the role of CTL cytolysis in immunity to intracellular pathogens.
Induction of Specific Immune Responses by Polycation-Based Vaccines
Authors: Lührs Petra , Schmidt Walter , Kutil Raphaela , Buschle Michael , Wagner Stephan N. , Stingl Georg , Schneeberger Achim
Published: April 2014

The s.c injection of tumor Ag-derived, MHC class I-binding peptides together with cationic poly-amino acids (e.g., poly-l-arginine; pR) has been shown to protect animals against a challenge with tumor cells expressing the respective peptide(s). Given our only restricted knowledge about immunogenic tumor-associated peptides, we sought to determine whether this pR-based vaccination protocol would also induce protective cancer immunity if large proteins were used instead of peptide epitopes. We found that the intracutaneous administration of the model Ag β-galactosidase (β-gal) together with pR (referred to as pR-based protein vaccine; pR-PV) was significantly more potent in protecting mice against the growth of β-gal-expressing RENCA cells than the protein alone. Coadministration of pR enhanced both the β-gal-induced specific humoral and CD8 response. The protective effect required CD8+, but neither CD4+ T lymphocytes nor β-gal-specific Abs. β-Gal priming of protective CD8+ T lymphocytes was found to be CD4+ T cell-independent, to take place within the draining lymph nodes, and to be accomplished by day 5 after vaccination. Ablation of the injection sites as early as 1.5 h after pR-PV administration still led to protection in a large proportion of the animals, indicating that certain protein Ags administered intradermally in the context of polycations are quickly transported to the draining nodes, where they induce molecular and cellular events resulting in the helper-independent priming and expansion of Tc1 cells. However, optimal protection required the prolonged presence of the injection site, suggesting that pR-PV injection facilitates the formation of a cutaneous depot of Ag-charged cells capable of migration and T cell activation.
Selective Suppression of Neutrophil Accumulation in Ongoing Pulmonary Inflammation by Systemic Inhibition of p38 Mitogen-Activated Protein Kinase
Authors: Nick Jerry A. , Young Scott K. , Arndt Patrick G. , Lieber Jonathan G. , Suratt Benjamin T. , Poch Katie R. , Avdi Natalie J. , Malcolm Ken C. , Taube Christian , Henson Peter M. , Worthen G. Scott
Published: April 2014

The p38 mitogen-activated protein kinase (MAPK) signaling pathway regulates a wide range of inflammatory responses in many different cells. Inhibition of p38 MAPK before exposing a cell to stress stimuli has profound anti-inflammatory effects, but little is known about the effects of p38 MAPK inhibition on ongoing inflammatory responses. LPS-induced activation of p38 MAPK in human neutrophils was inhibited by poststimulation exposure to a p38 MAPK inhibitor (M39). Release of TNF-α, macrophage-inflammatory protein (MIP)-2 (MIP-1β), and IL-8 by LPS-stimulated neutrophils was also reduced by poststimulation p38 MAPK inhibition. In contrast, release of monocyte chemoattractant protein-1 was found to be p38 MAPK independent. Ongoing chemotaxis toward IL-8 was eliminated by p38 MAPK inhibition, although the rate of nondirectional movement was not reduced. A murine model of acute LPS-induced lung inflammation was used to study the effect of p38 MAPK inhibition in ongoing pulmonary inflammation. Initial pulmonary cell responses occur within 4 h of stimulation in this model, so M39 was administered 4 h or 12 h after exposure of the animals to aerosolized LPS to avoid inhibition of cytokine release. Quantities of TNF-α, MIP-2, KC, or monocyte chemoattractant protein-1 recovered from bronchial alveolar lavage or serum were not changed. Recruitment of neutrophils, but not other leukocytes, to the airspaces was significantly reduced. Together, these data demonstrate the selective reduction of LPS-induced neutrophil recruitment to the airspaces, independent of suppression of other inflammatory responses. These findings support the feasibility of p38 MAPK inhibition as a selective intervention to reduce neutrophilic inflammation.
A Small Molecule Inhibitor of Redox-Regulated NF-κB and Activator Protein-1 Transcription Blocks Allergic Airway Inflammation in a Mouse Asthma Model
Authors: Henderson William R. , Chi Emil Y. , Teo Jia-Ling , Nguyen Cu , Kahn Michael
Published: April 2014

An oxidant/antioxidant imbalance is seen in the lungs of patients with asthma. This oxidative stress in asthmatic airways may lead to activation of redox-sensitive transcription factors, NF-κB and AP-1. We examined the effect of the small molecule inhibitor of redox-regulated NF-κB and AP-1 transcription, MOL 294 on airway inflammation and airway hyperreactivity (AHR) in a mouse model of asthma. MOL 294 is a potent nonpeptide inhibitor of NF-κB and AP-1 based upon a β-strand template that binds to and inhibits the cellular redox protein thioredoxin. BALB/c mice after i.p. OVA sensitization (day 0) were challenged with intranasal OVA on days 14, 25, 26, and 27. MOL 294, administered intranasal on days 25–27, blocked the airway inflammatory response to OVA assessed 24 h after the last OVA challenge on day 28. MOL 294 reduced eosinophil, IL-13, and eotaxin levels in bronchoalveolar lavage fluid and airway tissue eosinophilia and mucus hypersecretion. MOL 294 also decreased AHR in vivo to methacholine. These results support redox-regulated transcription as a therapeutic target in asthma and demonstrate that selective inhibitors can reduce allergic airway inflammation and AHR.
Protease-Activated Receptor 2 Mediates Eosinophil Infiltration and Hyperreactivity in Allergic Inflammation of the Airway
Authors: Schmidlin Fabien , Amadesi Silvia , Dabbagh Karim , Lewis David E. , Knott Patrick , Bunnett Nigel W. , Gater Paul R. , Geppetti Pierangelo , Bertrand Claude , Stevens Mary E.
Published: April 2014

Trypsin and mast cell tryptase can signal to epithelial cells, myocytes, and nerve fibers of the respiratory tract by cleaving proteinase-activated receptor 2 (PAR2). Since tryptase inhibitors are under development to treat asthma, a precise understanding of the contribution of PAR2 to airway inflammation is required. We examined the role of PAR2 in allergic inflammation of the airway by comparing OVA-sensitized and -challenged mice lacking or overexpressing PAR2. In wild-type mice, immunoreactive PAR2 was detected in airway epithelial cells and myocytes, and intranasal administration of a PAR2 agonist stimulated macrophage infiltration into bronchoalveolar lavage fluid. OVA challenge of immunized wild-type mice stimulated infiltration of leukocytes into bronchoalveolar lavage and induced airway hyperreactivity to inhaled methacholine. Compared with wild-type animals, eosinophil infiltration was inhibited by 73% in mice lacking PAR2 and increased by 88% in mice overexpressing PAR2. Similarly, compared with wild-type animals, airway hyperreactivity to inhaled methacholine (40 μg/ml) was diminished 38% in mice lacking PAR2 and increased by 52% in mice overexpressing PAR2. PAR2 deletion also reduced IgE levels to OVA sensitization by 4-fold compared with those of wild-type animals. Thus, PAR2 contributes to the development of immunity and to allergic inflammation of the airway. Our results support the proposal that tryptase inhibitors and PAR2 antagonists may be useful therapies for inflammatory airway disease.
Prolonged Maturation and Enhanced Transduction of Dendritic Cells Migrated from Human Skin Explants After In Situ Delivery of CD40-Targeted Adenoviral Vectors
Authors: de Gruijl Tanja D. , Luykx-de Bakker Sylvia A. , Tillman Bryan W. , van den Eertwegh Alfons J. M. , Buter Jan , Lougheed Sinéad M. , van der Bij Gerben J. , Safer A. Mahmoud , Haisma Hidde J. , Curiel David T. , Scheper Rik J. , Pinedo Herbert M. , Gerritsen Winald R.
Published: April 2014

Therapeutic tumor vaccination with viral vectors or naked DNA, carrying the genetic code for tumor-associated Ags, critically depends on the in vivo transduction of dendritic cells (DC). Transfection of predominantly nonprofessional APC and only small numbers of DC may hamper proper T cell activation. Aim of this study was, therefore, the targeted, selective, and enhanced in situ transduction of DC. A human skin explant model was used to explore targeted transduction of cutaneous DC after intradermal injection of a bispecific Ab conjugate to link adenoviral (Ad) vectors directly to CD40 on the DC surface. A significantly enhanced transduction efficiency and selectivity, and an increased activation state of migrating DC were thus achieved. Moreover, DC transduced by CD40-targeted Ad maintained their Ag-specific CTL-stimulatory ability for up to 1 wk after the start of migration, in contrast to DC transduced by untargeted Ad, which had lost this capacity by that time. Because DC targeting in vivo might obviate the need for the in vitro culture of autologous DC for adoptive transfer, CD40-targeted Ad vectors constitute a promising new vaccine modality for tumor immunotherapy.
Dendritic Cells Process and Present Antigens Across A Range of Maturation States
Authors: Veeraswamy Ravi K. , Cella Marina , Colonna Marco , Unanue Emil R.
Published: April 2014

We isolated dendritic cells (DC) from lymphoid organs of mice bearing a transgene for a membrane-bound form of the model protein hen egg white lysozyme (HEL). DC from the spleen had a lower representation of costimulatory molecules and class II MHC molecules than those isolated from lymph nodes and thymi. Splenic DC were capable of further maturation by in vivo treatment of mice with LPS. The immature DC from spleen processed HEL and displayed the chemically dominant epitope as evidenced by FACS analysis. These immature DC also presented this epitope to CD4+ T cells. Splenic DC from another transgenic mouse (ML-5) containing serum HEL also showed the ability to process and present Ag despite low levels of circulating HEL. In vitro-derived DC from the bone marrow (bone marrow-derived DC) of mHEL mice also displayed immature to mature features and in both cases displayed HEL peptides as well as SDS-stable MHC class II molecules. Immature bone marrow-derived DC also processed exogenous HEL. We conclude that the DC sets normally found in tissue show a scale of maturation features but even the most immature process and present peptides by MHC class II molecules.
Generation of Cytotoxic Responses in Mice and Human Individuals Against Hematological Malignancies Using Survivin-RNA-Transfected Dendritic Cells
Authors: Zeis Matthias , Siegel Sandra , Wagner Andreas , Schmitz Marc , Marget Matthias , Kühl-Burmeister Rita , Adamzik Ilse , Kabelitz Dieter , Dreger Peter , Schmitz Norbert , Heiser Axel
Published: April 2014

Survivin is a member of the inhibitors of apoptosis family and is overexpressed in many types of human cancers, making it an attractive target for T cell-based immunotherapeutic strategies. Recently, HLA-A2-binding peptides derived from the survivin protein were identified as capable of inducing specific T cell responses in cancer patients. Here we demonstrate that human survivin-specific CTLs generated from PBMC by stimulation with autologous dendritic cells transfected with survivin-RNA were cytotoxic for a range of hemopoietic malignant cell lines and primary tumor cells isolated from patients with acute myeloid leukemia. We also show that vaccination of mice with survivin-RNA-transfected dendritic cells leads to long term resistance to challenge by a survivin-expressing lymphoma, demonstrating the potential of survivin as a tumor rejection Ag. Our data provide evidence for the use of survivin as a target structure for immunotherapeutic strategies against hematological neoplasms.
A Role for the B7-1/B7-2:CD28/CTLA-4 Pathway During Negative Selection
Authors: Buhlmann Janet E. , Elkin Sheryl Krevsky , Sharpe Arlene H.
Published: April 2014

Although costimulation plays an important role in activating naive T cells, its role in negative selection is controversial. By following thymocyte deletion induced by endogenous superantigens in mice lacking B7-1 and/or B7-2, we have identified a role for both B7-1 and B7-2 in negative selection. Studies using CD28-deficient and CD28/CTLA-4-double-deficient mice have revealed that either CD28 or another as yet undefined coreceptor can mediate these B7-dependent signals that promote negative selection. Finally, CTLA-4 delivers signals that inhibit selection, suggesting that CTLA-4 and CD28 have opposing functions in thymic development. Combined, the data demonstrate that B7-1/B7-2-dependent signals help shape the T cell repertoire.
TCR Signal Transduction in Antigen-Specific Memory CD8 T Cells
Authors: Kersh Ellen N. , Kaech Susan M. , Onami Thandi M. , Moran Miriana , Wherry E. John , Miceli M. Carrie , Ahmed Rafi
Published: April 2014

Memory T cells are more responsive to Ag than naive cells. To determine whether memory T cells also have more efficient TCR signaling, we compared naive, effector, and memory CD8 T cells of the same antigenic specificity. Surprisingly, initial CD3 signaling events are indistinguishable. However, memory T cells have more extensive lipid rafts with higher phosphoprotein content before TCR engagement. Upon activation in vivo, they more efficiently induce phosphorylation of-LAT (linker for activation of T cells), ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), and p38. Thus, memory CD8 T cells do not increase their TCR sensitivity, but are better poised to augment downstream signals. We propose that this regulatory mechanism might increase signal transduction in memory T cells, while limiting TCR cross-reactivity and autoimmunity.
Lung CD25 CD4 Regulatory T Cells Suppress Type 2 Immune Responses But Not Bronchial Hyperreactivity
Authors: Hadeiba Husein , Locksley Richard M.
Published: April 2014

To study the effects of chronic Ag deposition in the airway mucosa on CD4+ T cell priming and subsequent airway disease, transgenic mice were generated that expressed OVA under the control of the surfactant protein C promoter. CD4 T cells from these mice were tolerant to OVA but this was overcome among spleen CD4 T cells by crossing to OVA-specific DO11.10 TCR-transgenic mice. Lungs from the double-transgenic mice developed lymphocytic infiltrates and modest mucus cell hyperplasia. Infiltrating cells were unaffected by the absence of either Rag-1 or Stat6, although the latter deficiency led to the disappearance of mucus. In the lung of double-transgenic mice, a large number of Ag-specific CD4 T cells expressed CD25 and functioned as regulatory T cells. The CD25+ CD4 T cells suppressed proliferation of CD25− CD4 T cells in vitro and inhibited type 2 immune responses induced by aerosolized Ags in vivo. Despite their ability to suppress allergic type 2 immunity in the airways, however, CD25+ CD4 regulatory T cells had no effect on the development of bronchial hyperreactivity.
Notch Signaling Induces Multilineage Myeloid Differentiation and Up-Regulates PU.1 Expression
Authors: Schroeder Timm , Kohlhof Hella , Rieber Nikolaus , Just Ursula
Published: April 2014

Hemopoietic commitment is initiated by and depends on activation of transcription factors. However, it is unclear whether activation of lineage-affiliated transcription factors is extrinsically regulated by to date unknown agents or is the result of a cell autonomous program. Here we show that signaling by the Notch1 transmembrane receptor instructively induces myeloid differentiation of multipotent hemopoietic progenitor cells and concomitantly up-regulates the expression of the transcription factor PU.1. Transient activation of Notch1 signaling is sufficient to irreversibly reduce self-renewal of multipotent progenitor cells accompanied by increased and accelerated differentiation along the granulocyte, macrophage, and dendritic cell lineages. Activated Notch1 has no direct influence on apoptosis of multipotent progenitor cells, shows a weak inhibition of proliferation, and does not substitute for survival and proliferation signals provided by cytokines. Activated Notch1 directly increases PU.1 RNA levels, leading to a high concentration of PU.1 protein, which has been shown to direct myeloid differentiation. These findings identify Notch as an extrinsic regulator of myeloid commitment, and the lineage-affiliated transcription factor PU.1 as a specific direct target gene of Notch.
Yin Yang 1 Is a Lipopolysaccharide-Inducible Activator of the Murine 3′IghEnhancer, hs3
Authors: Gordon Steven J. , Saleque Shireen , Birshtein Barbara K.
Published: April 2014

The 3′ Igh enhancers, DNase I hypersensitive site (hs) 3B and/or hs4, are required for germline transcription, and hence, class switch recombination for multiple isotypes. A number of hs3-binding transcription factors have been identified by EMSA, including octamer and NF-κB family members, and Pax5. We have found that the binding of the transcription factor, Yin Yang 1 (YY1), to hs3 and to the μE1 site of the intronic enhancer, Eμ, is induced in primary splenic B cells after ∼48 h in response to LPS and other activators of class switch recombination. Transient transfection experiments in B cell lines indicate that YY1 is an activator of hs3. Interestingly, levels of YY1 expression are unchanged in resting and LPS-stimulated B cells. Mixing experiments followed by EMSA showed that a protein present in resting B cells prevented binding of YY1 to DNA. We found that recombinant retinoblastoma protein (Rb) inhibited binding of YY1 to hs3 in a dose-dependent manner, and we have identified complexes of endogenous YY1 with the Rb in resting B cells, but not in LPS-stimulated B cells. A difference in Rb phosphorylation state was also confirmed between resting (G0) B cells and LPS-stimulated B cells. These observations suggest that the interaction of YY1 with hypophosphorylated Rb in resting B cells prevents interaction of YY1 with DNA. After stimulation with class-switching activators, such as LPS, Rb becomes hyperphosphorylated and YY1 is released and can then bind to the hs3 enhancer and Eμ.
Uncoupling of Promitogenic and Antiapoptotic Functions of IL-2 by Smad-Dependent TGF-β Signaling
Authors: Nelson Brad H. , Martyak Timothy P. , Thompson Lucas J. , Moon James J. , Wang Tongwen
Published: April 2014

TGF-β opposes proliferative signaling by IL-2 through mechanisms that remain incompletely defined. In a well-characterized CD8+ T cell model using wild-type and mutated IL-2 receptors, we examined the effects of TGF-β on distinct IL-2 signaling events in CD8+ T cells. IL-2 induces c-myc, cyclin D2, and cyclin E in a redundant manner through the Shc and STAT5 pathways. TGF-β inhibited the ability of either the Shc or STAT5 pathway to induce these genes, as well as T cell proliferation. The inhibitory effects of TGF-β were reversed by expression of a dominant-negative form of Smad3. TGF-β did not impair proximal signaling by Shc or STAT5, and induction of some downstream genes, including cytokine-inducible Src homology-2-containing protein (CIS), bcl-xL, and bcl-2, was spared. Experiments with c-fos, cyclin D2, and CIS reporter genes revealed that promoter-proximal regulatory elements dictate the sensitivity of IL-2 target genes to inhibition by TGF-β. By leaving the Shc and STAT5 pathways functional while inhibiting their target genes selectively, TGF-β was found to uncouple the proliferative and antiapoptotic functions of IL-2. Thus, TGF-β is not a simple antagonist of IL-2, but rather serves to qualitatively modify the IL-2 signal to create a unique pattern of gene expression that neither cytokine can induce independently.
IL-18 Receptor β-Induced Changes in the Presentation of IL-18 Binding Sites Affect Ligand Binding and Signal Transduction
Authors: Wu Chengbin , Sakorafas Paul , Miller Renee , McCarthy Donna , Scesney Susanne , Dixon Richard , Ghayur Tariq
Published: April 2014

IL-18 is a pleiotropic proinflammatory cytokine that is involved in induction of inflammatory mediators, regulation of the cytotoxic activity of NK cells and T cells, and differentiation and activation of both Th1 and Th2 cells. IL-18 signals through its specific cell surface receptor IL-18R, which comprises two subunits: IL-18Rα and IL-18Rβ. IL-18Rα alone has a weak affinity for IL-18 binding, while the IL-18Rα/β complex has a high affinity. By using several anti-IL-18 mAbs and IL-18 binding protein, we have examined whether these site-specific inhibitors could block the binding of IL-18 to IL-18Rα and to the IL-18Rα/β complex. Here we show that IL-18 binding to IL-18Rα was inhibited by a neutralizing mAb, 125-2H, while binding of IL-18 to the α/β receptor complex was not. This suggests that IL-18Rβ-induced conformational changes may occur in IL-18Rα upon dimerization, leading to changes in the presentation of IL-18 binding sites. Epitope mapping of 125-2H using human-mouse IL-18 chimeras identified a region in IL-18 that was required for 125-2H recognition. This region, as examined by IL-18R binding and functional analysis, appeared to be critical for triggering signal transduction through the heterodimeric receptor.
Renewal of Peripheral CD8+ Memory T Cells During Secondary Viral Infection of Antibody-Sufficient Mice
Authors: Cauley Linda S. , Cookenham Tres , Hogan Robert J. , Crowe Sherry R. , Woodland David L.
Published: April 2014

Kinetic studies and short pulses of injected 5-bromo-2-deoxyuridine have been used to analyze the development and renewal of peripheral CD8+ memory T cells in the lungs during primary and secondary respiratory virus infections. We show that developing peripheral CD8+ memory T cells proliferate during acute viral infection with kinetics that are indistinguishable from those of lymphoid CD8+ memory T cells. Secondary exposure to the same virus induces a new round of T cell proliferation and extensive renewal of the peripheral and lymphoid CD8+ memory T cell pools in both B cell-deficient mice and mice with immune Abs. In mice with virus-specific Abs, CD8+ T cell proliferation takes place with minimal inflammation or effector cell recruitment to the lungs. The delayed arrival of CD8+ memory T cells to the lungs of these animals suggests that developing memory cells do not require the same inflammatory signals as effector cells to reach the lung airways. These studies provide important new insight into mechanisms that control the maintenance and renewal of peripheral memory T cell populations during natural infections.
Neutrophil Serine Proteinases Activate Human Nonepithelial Cells to Produce Inflammatory Cytokines Through Protease-Activated Receptor 2
Authors: Uehara Akiko , Muramoto Koji , Takada Haruhiko , Sugawara Shunji
Published: April 2014

Protease-activated receptors (PARs) compose a family of G protein-coupled receptors activated by proteolysis with exposure of their tethered ligand. Recently, we reported that a neutrophil-derived serine proteinase, proteinase 3 (PR3), activated human oral epithelial cells through PAR-2. The present study examined whether other neutrophil serine proteinases, human leukocyte elastase (HLE), and cathepsin G (Cat G) activate nonepithelial cells, human gingival fibroblasts (HGF). HLE and Cat G as well as PR3 activated HGF to produce IL-8 and monocyte chemoattractant protein 1. Human oral epithelial cells but not HGF express mRNA and protein of secretory leukocyte protease inhibitor, an inhibitor of HLE and Cat G, and recombinant secretory leukocyte protease inhibitor clearly inhibited the activation of HGF induced by HLE and Cat G but not by PR3. HGF express PAR-1 and PAR-2 mRNA in the cells and the proteins on the cell surface. HLE and Cat G cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca2+ mobilization and rendered cells refractory to subsequent stimulation with HLE and Cat G. The production of cytokine induced by HLE and Cat G and the PAR-2 agonist peptide was completely abolished by inhibition of phospholipase C. These findings suggest that neutrophil serine proteinases have equal ability to activate human nonepithelial cells through PAR-2 to produce inflammatory cytokines and may control a number of inflammatory processes such as periodontitis.
Synergistic Effect of TNF-α in Soluble VCAM-1-Induced Angiogenesis Through α4 Integrins
Authors: Nakao Shintaro , Kuwano Takashi , Ishibashi Tatsuro , Kuwano Michihiko , Ono Mayumi
Published: April 2014

In our present study we focused on soluble VCAM-1 (sVCAM-1)/α4 integrin-induced angiogenesis and found that this type of angiogenesis was mediated through p38 mitogen-activated protein kinase and focal adhesion kinase (FAK). HUVEC expressed both α4 and β1 integrins, and it was reported that expression of α4 integrin and its counterreceptor, sVCAM-1/VCAM-1, was enhanced in response to an inflammatory cytokine, TNF-α. In endothelial cells phosphorylation of p38 and FAK, but not that of extracellular signal-regulated kinase 1/2 was induced by sVCAM-1. Migration of endothelial cells was stimulated in response to sVCAM-1 at similar levels as those induced by vascular endothelial growth factor, and sVCAM-1-induced migration was almost completely blocked by neutralizing Ab against α4 integrin, by either an inhibitor of p38 (SB203580), or by adenovirus containing FAK-related nonkinase. sVCAM-1 also induced the formation of blood vessels in Matrigel plug assay in vivo, and this neovascularization was blocked by SB203580 or neutralizing Ab against α4 integrin. Moreover, we also confirmed that both TNF-α and sVCAM-1 could synergistically induce angiogenesis in the corneas of mice when each factor at used dose could not induce. This angiogenesis by TNF-α and sVCAM-1 was almost completely blocked by coadministration of SB203580 and also by neutralizing Ab against α4 integrin. These results suggest that sVCAM-1/α4 integrin induces angiogenesis through p38 and FAK signaling pathways.
T Cells of Atopic Asthmatics Preferentially Infiltrate Into Human Bronchial Xenografts in SCID Mice
Authors: Tsumori Kaori , Kohrogi Hirotsugu , Goto Eisuke , Hirata Naomi , Hirosako Susumu , Fujii Kazuhiko , Ando Makoto , Kawano Osamu , Mizuta Hiroshi
Published: April 2014

T cells play an important role in the pathogenesis of bronchial asthma. However, it is not completely known how circulating lymphocytes infiltrate into the airways of asthmatic patients. Because SCID mice are unable to reject xenogenic transplants, many xenotransplant models using various human tissues have been developed. Therefore, to examine the interaction between bronchi and T lymphocytes of asthma, it may be possible to use the human bronchial xenograft and PBMC xenograft in SCID mice. We transplanted human bronchi into the subcutaneum of SCID mice and i.p. injected PBMCs that were obtained from patients with atopic asthma, atopic dermatitis and rheumatoid arthritis, and normal subjects (asthmatic, dermatitis, rheumatic, and normal huPBMC-SCID mice). There was no difference in the percentage of CD3-, CD4-, CD8-, CD25-, CD45RO-, CD103-, and cutaneous lymphocyte Ag-positive cells in PBMCs among the patients with asthma, dermatitis, rheumatoid arthritis, and normal subjects, and CD3-positive cells in peripheral blood of asthmatic, dermatitis, rheumatic, and normal huPBMC-SCID mice. The number of CD3-, CD4-, and CD8-positive cells in the xenografts of asthmatic huPBMC-SCID mice was higher than those of dermatitis, rheumatic, and normal huPBMC-SCID mice. IL-4 mRNA and IL-5 mRNA were significantly higher in the xenografts of asthmatic huPBMC-SCID mice than those in the xenografts of normal huPBMC-SCID mice, but there were no significant differences in the expressions of IL-2 mRNA or IFN-γ mRNA between them. These findings suggest that T cells, especially Th2-type T cells, of asthmatics preferentially infiltrate into the human bronchi.
HIV-1 Replication Increases HIV-Specific CD4+ T Cell Frequencies but Limits Proliferative Capacity in Chronically Infected Children
Authors: Scott Zachary A. , Beaumier Coreen M. , Sharkey Mark , Stevenson Mario , Luzuriaga Katherine
Published: April 2014

This study investigated the relationship between HIV-1 replication and virus (HIV-1; CMV)-specific CD4+ T cell frequency and function in HIV-1-infected children. HIV-1 gag p55-specific CD4+ T cell IFN-γ responses were detected in the majority of children studied. p55-specific responses were detected less commonly and at lower frequencies in children with <50 copies/ml plasma HIV-1 RNA than in children with active HIV-1 replication. In children with <50 copies/ml plasma HIV-1, p55-specific responses were detected only in children with evidence of ongoing HIV-1 replication, indicating a direct relationship between HIV-1 replication and HIV-specific CD4+ T cell frequencies. In contrast, p55-specific proliferative responses were detected more frequently in children with <50 copies/ml plasma HIV-1. CMV-specific CD4+ responses were more commonly detected and at higher frequencies in CMV-coinfected children with suppressed HIV-1 replication. The lack of HIV-specific CD4+ proliferative responses, along with the preservation of CMV-specific CD4+ responses in children with controlled HIV-1 replication, suggests that viral replication may have deleterious effects on HIV-1 and other virus-specific CD4+ responses. Vaccination to stimulate HIV-specific CD4+ T cell responses in these children may synergize with antiretroviral therapy to improve the long-term control of viral replication, and may perhaps allow the eventual discontinuation of antiretroviral therapy.
Regulatory T Cells Secreting IL-10 Dominate the Immune Response to EBV Latent Membrane Protein 1
Authors: Marshall Neil A. , Vickers Mark A. , Barker Robert N.
Published: April 2014

Viruses exploit a number of strategies to evade immune recognition. In this study, we describe a novel mechanism by which EBV, rather than avoiding detection, subverts the immune response by stimulating regulatory T cells that secrete IL-10. Human PBMC from all EBV-seropositive, but not -seronegative, donors responded to both purified latent membrane protein 1 and the corresponding immunodominant peptides with high levels of IL-10 secretion by CD4+ T cells. These IL-10 responses, characteristic of T regulatory 1 cells, inhibited T cell proliferation and IFN-γ secretion induced by both mitogen and recall Ag. It was confirmed that the inhibition was IL-10 dependent by the use of neutralizing Ab. The deviation of the immune response toward suppression is likely to be important in maintaining latency and EBV-associated tumors.
Fas (CD95) Induces Proinflammatory Cytokine Responses by Human Monocytes and Monocyte-Derived Macrophages
Authors: Park David R. , Thomsen Anni R. , Frevert Charles W. , Pham Uyenvy , Skerrett Shawn J. , Kiener Peter A. , Liles W. Conrad
Published: April 2014

Fas (CD95, APO-1) is regarded as the prototypical cell death receptor of the TNFR superfamily. Fas-induced apoptosis is generally considered to be a noninflammatory process, contributing to the silent resolution of immune and inflammatory responses. However, accumulating evidence indicates that Fas may also induce cellular activation signals. We hypothesized that Fas could activate proinflammatory cytokine responses by normal human monocytes and macrophages. Monocytes were isolated by negative immunoselection from the PBMC fraction of venous blood from healthy volunteers, and monocyte-derived macrophages were cultivated in vitro. Both monocytes and monocyte-derived macrophages released TNF-α and IL-8 following Fas ligation, and conditioned medium from Fas-activated monocytes and macrophages induced the directed migration of neutrophils in a chemotaxis assay. Fas-induced monocyte cytokine responses were associated with monocyte apoptosis, nuclear translocation of NF-κB, and cytokine gene expression and were blocked by caspase inhibition but not by inhibition of IL-1β signaling. In contrast, Fas-induced macrophage cytokine responses occurred in the absence of apoptosis and were caspase independent, indicating maturation-dependent differences in the Fas signaling pathways that lead to proinflammatory cytokine induction. Rather than contributing to the resolution of inflammation, Fas ligation on circulating monocytes and tissue macrophages may induce proinflammatory cytokine responses that can initiate acute inflammatory responses and tissue injury.
Novel Quantitative Trait Loci Controlling Development of Experimental Autoimmune Encephalomyelitis and Proportion of Lymphocyte Subpopulations
Authors: Karlsson Jenny , Zhao Xiangshan , Lonskaya Irina , Neptin Malin , Holmdahl Rikard , Andersson Åsa
Published: April 2014

The B10.RIII mouse strain (H-2r) develops chronic experimental autoimmune encephalomyelitis (EAE) upon immunization with the myelin basic protein 89–101 peptide. EAE was induced and studied in a backcross between B10.RIII and the EAE-resistant RIIIS/J strain (H-2r), and a complete genome scan with microsatellite markers was performed. Five loci were significantly linked to different traits and clinical subtypes of EAE on chromosomes 1, 5, 11, 15, and 16, three of the loci having sex specificity. The quantitative trait locus on chromosome 15 partly overlapped with the Eae2 locus, previously identified in crosses between the B10.RIII and RIIIS/J mouse strains. The loci on chromosomes 11 and 16 overlapped with Eae loci identified in other mouse crosses. By analyzing the backcross animals for lymphocyte phenotypes, the proportion of B and T cells in addition to the levels of CD4+CD8− and CD4−CD8+ T cells and the CD4+/CD8+ ratio in spleen were linked to different loci on chromosomes 1, 2, 3, 5, 6, 11, and 15. On chromosome 16, we found significant linkage to spleen cell proliferation. Several linkages overlapped with the quantitative trait loci for disease phenotypes. The identification of subphenotypes that are linked to the same loci as disease traits could be most useful in the search for candidate genes and biological pathways involved in the pathological process.
Cyclooxygenase-2 Expression by Nonsteroidal Anti-inflammatory Drugs in Human Airway Smooth Muscle Cells: Role of Peroxisome Proliferator-Activated Receptors
Authors: Pang Linhua , Nie Mei , Corbett Lisa , Knox Alan J.
Published: April 2014

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to modulate cyclooxygenase (COX)-2 expression, but the mechanisms involved are controversial and may be cell specific. We show in this study that indomethacin (Indo), flurbiprofen (Flur), and the selective COX-2 inhibitor NS-398 induced COX-2 expression and markedly enhanced IL-1β-induced COX-2 expression in human airway smooth muscle (HASM) cells. These effects were not reversed by exogenous PGE2, suggesting that they are prostanoid-independent. Indeed, PGE2 also induced and enhanced IL-1β-induced COX-2 expression. Peroxisome proliferator-activated receptor (PPAR) α and PPARγ (not PPARβ) were expressed in HASM cells. PPARγ activators ciglitizone (Cig) and 15-Deoxy-Δ12,14-PGJ2 (15d-PGJ2), but not the PPARα activator WY-14643, mimicked the effect of NSAIDs on COX-2 expression. Treatment with Flur, NS-398, Cig, and 15d-PGJ2 alone, but not Indo and WY-14643, elevated COX activity; however, neither enhanced IL-1β-induced COX activity. Pretreatment with dexamethasone suppressed COX-2 expression, PGE2 release, and COX activity induced by NS-398, Cig, IL-1β, alone or in combination. Unlike IL-1β, NS-398 and Cig did not cause NF-κB (p65) nuclear translocation, nor did they further enhance IL-1β-induced NF-κB translocation, but they stimulated PPARγ translocation. Indo, NS-398, Flur, and 15d-PGJ2, but not WY-14643, induced transcriptional activity of a COX-2 reporter construct containing the peroxisome proliferator response element (PPRE) on their own and enhanced the effect of IL-1β, but had no effect on a COX-2 reporter construct lacking the PPRE. The results suggest that COX-2 expression by NSAIDs is biologically functional, prostanoid-independent, and involves PPARγ activation, and provide the first direct evidence that the PPRE in the promoter is required for NSAID-induced COX-2 expression.