bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Novel Murine CD4+CD8+ Thymocyte Cell Line Exhibits Lineage Commitment into Both CD4+ and CD8+ T Cells by Altering the Intensity and the Duration of Anti-CD3 Stimulation In Vitro
Authors: Nishida Takashi , Matsuki Yasunori , Ono Takeshi , Oguma Takemi , Tsujimoto Kyoko , Sato Masaki , Tadakuma Takushi 
Published: April 2014

Abstract
A CD4+CD8+ double-positive thymocyte cell line, 257-20-109 was established from BALB/c mice thymocytes and used to analyze the requirements to induce CD4 or CD8 single-positive (SP) T cells. CD4SP cells were induced from 257-20-109 cells by anti-CD3 stimulation in the presence of the FcR-positive macrophage cell line, P388D1. During stimulation, maturation events, such as the down-regulation of CD24 and the up-regulation of CD69, H-2Dd, CD5, and Bcl-2, were recognized. Furthermore, these CD4SP cells appeared to be functional because the cells produced IL-2 and IL-4 when activated with phorbol ester and calcium ionophore. In contrast, CD8SP cells could be induced by stimulation with fixed anti-CD3 after removal of stimulation. To investigate the extent of signals required for CD4SP and CD8SP, the cells stimulated under either condition for 2 days were sorted and transferred to different culture conditions. These results suggested that the fate of lineage commitment was determined within 2 days, and that CD4 lineage commitment required longer activation. Furthermore, the experiments with subclones of 257-20-109 demonstrated that the lower density of CD3 did not shift the cells from CD4SP to CD8SP, but only reduced the amount of CD4SP cells. In contrast, when the 257-20-109 cells were stimulated by the combination of fixed anti-CD3 and anti-CD28, the majority of the cells shifted to CD4SP, with an enhancement of extracellular signal-regulated kinase 1 phosphorylation. Our results indicate that the signals via TCR/CD3 alone shifted the double-positive cells to CD8SP cells, but the reinforced signals via TCR/CD3 and costimulator could commit the cells to CD4SP.
A Novel Approach to Specific Allergy Treatment: The Recombinant Fusion Protein of a Bacterial Cell Surface (S-Layer) Protein and the Major Birch Pollen Allergen Bet v 1 (rSbsC-Bet v 1) Combines Reduced Allergenicity with Immunomodulating Capacity
Authors: Bohle Barbara , Breitwieser Andreas , Zwölfer Bettina , Jahn-Schmid Beatrice , Sára Margit , Sleytr Uwe B. , Ebner Christof 
Published: April 2014

Abstract
Counterregulating the disease-eliciting Th2-like immune response of allergen-specific Th lymphocytes by fostering an allergen-specific Th1-like response is a promising concept for future immunotherapy of type I allergy. The use of recombinant allergens combined with more functional adjuvants has been proposed. In this respect, we present a novel approach. The gene sequence encoding the major birch pollen allergen, Bet v 1, was fused with the gene encoding the bacterial cell surface (S-layer) protein of Geobacillus stearothermophilus, resulting in the recombinant protein, rSbsC-Bet v 1. rSbsC-Bet v 1 contained all relevant Bet v 1-specific B and T cell epitopes, but was significantly less efficient to release histamine than rBet v 1. In cells of birch pollen-allergic individuals, rSbsC-Bet v 1 induced IFN-γ along with IL-10, but no Th2-like response, as observed after stimulation with Bet v 1. Intracellular cytokine staining revealed that rSbsC-Bet v 1 promoted IFN-γ-producing Th cells. Moreover, rSbsC-Bet v 1 induced IFN-γ synthesis in Bet v 1-specific Th2 cell clones, and importantly, increased IL-10 production in these cells. In conclusion, genetic fusion of an allergen to S-layer proteins combined reduced allergenicity with immunomodulatory capacity. The strategy described in this work may be generally applied to design vaccines for specific immunotherapy of type I allergy with improved efficacy and safety.
Messenger RNA-Electroporated Dendritic Cells Presenting MAGE-A3 Simultaneously in HLA Class I and Class II Molecules
Authors: Bonehill Aude , Heirman Carlo , Tuyaerts Sandra , Michiels Annelies , Breckpot Karine , Brasseur Francis , Zhang Yi , van der Bruggen Pierre , Thielemans Kris 
Published: April 2014

Abstract
An optimal anticancer vaccine probably requires the cooperation of both CD4+ Th cells and CD8+ CTLs. A promising tool in cancer immunotherapy is, therefore, the genetic modification of dendritic cells (DCs) by introducing the coding region of a tumor Ag, of which the antigenic peptides will be presented in both HLA class I and class II molecules. This can be achieved by linking the tumor Ag to the HLA class II-targeting sequence of an endosomal or lysosomal protein. In this study we compared the efficiency of the targeting signals of invariant chain, lysosome-associated membrane protein-1 (LAMP1) and DC-LAMP. Human DCs were electroporated before or after maturation with mRNA encoding unmodified enhanced green fluorescent protein (eGFP) or eGFP linked to various targeting signals. The lysosomal degradation inhibitor chloroquine was added, and eGFP expression was evaluated at different time points after electroporation. DCs were also electroporated with unmodified MAGE-A3 or MAGE-A3 linked to the targeting signals, and the presentation of MAGE-A3-derived epitopes in the context of HLA class I and class II molecules was investigated. Our data suggest that proteins linked to the different targeting signals are targeted to the lysosomes and are indeed presented in the context of HLA class I and class II molecules, but with different efficiencies. Proteins linked to the LAMP1 or DC-LAMP signal are more efficiently presented than proteins linked to the invariant chain-targeting signal. Furthermore, DCs electroporated after maturation are more efficient in Ag presentation than DCs electroporated before maturation.
Elimination of an Immunodominant CD4+ T Cell Epitope in Human IFN-β Does Not Result in an In Vivo Response Directed at the Subdominant Epitope
Authors: Yeung V. Peter , Chang Judy , Miller Jeff , Barnett Christopher , Stickler Marcia , Harding Fiona A. 
Published: April 2014

Abstract
The BALB/cByJ mouse strain displays an immunodominant T cell response directed at the same CD4+ T cell epitope peptide region in human IFN-β, as detected in a human population-based assay. BALB/cByJ mice also recognize a second region of the protein with a lesser magnitude proliferative response. Critical residue testing of the immunodominant peptide showed that both BALB/cByJ mice and the human population response were dependent on an isoleucine residue at position 129. A variant IFN-β molecule was constructed containing the single amino acid modification, I129V, in the immunodominant epitope. The variant displayed 100% of control antiproliferation activity. Mice immunized with unmodified IFN-β responded weakly in vitro to the I129V variant. However, BALB/cByJ mice immunized with the I129V variant were unable to respond to either the I129V variant or the unmodified IFN-β molecule by either T cell proliferation or Ag-specific IgG1 Ab production. This demonstrates that a single amino acid change in an immunodominant epitope can eliminate an immune response to an otherwise intact therapeutic protein. The elimination of the immunodominant epitope response also eliminated the response to the subdominant epitope in the protein. Modifying functionally immunodominant T cell epitopes within proteins may obviate the need for additional subdominant epitope modifications.
Duration of Alloantigen Presentation and Avidity of T Cell Antigen Recognition Correlate with Immunodominance of CTL Response to Minor Histocompatibility Antigens
Authors: Yoshimura Yoshitaka , Yadav Rajwardhan , Christianson Gregory J. , Ajayi Wilfred U. , Roopenian Derry C. , Joyce Sebastian 
Published: April 2014

Abstract
CD8 T lymphocytes (CTL) responsive to immunodominant minor histocompatibility (minor H) Ags are thought to play a disproportionate role in allograft rejection in MHC-identical solid and bone marrow transplant settings. Although many studies have addressed the mechanisms underlying immunodominance in models of infectious diseases, cancer immunotherapy, and allograft immunity, key issues regarding the molecular basis of immunodominance remain poorly understood. In this study, we exploit the minor H Ag system to understand the relationship of the various biochemical parameters of Ag presentation and recognition to immunodominance. We show that the duration of individual minor H Ag presentation and the avidity of T cell Ag recognition influence the magnitude and, hence, the immunodominance of the CTL response to minor H Ags. These properties of CTL Ag presentation and recognition that contribute to immunodominance have implications not only for tissue transplantation, but also for autoimmunity and tumor vaccine design.
APCs in the Anterior Uveal Tract Do Not Migrate to Draining Lymph Nodes
Authors: Dullforce Per A. , Garman Kiera L. , Seitz Greg W. , Fleischmann Ross J. , Crespo Sergio M. , Planck Stephen R. , Parker David C. , Rosenbaum James T. 
Published: April 2014

Abstract
The migration of APCs from sites of infection and their maturation are critical elements in the generation of immune responses. However, the paths by which intraocular Ags migrate to draining lymph nodes are not known because the eye has limited lymphatic vessels. To date, only dendritic cells from the cornea and conjunctiva have been shown to emigrate. We demonstrate that phagocytic APCs in the anterior uveal tissues of the murine eye that ingest fluorescent latex beads do not migrate to regional lymph nodes. The beads are ingested in the uveal tract by cells expressing MHC class II, CD11c, or F4/80. Using intravital time-lapse videomicroscopy to monitor iris APC migration after anterior chamber injection of fluorescent Ag, fluorescently labeled APCs fail to move at multiple observation times, even in the presence of Ag and LPS. Whereas an as yet unidentified ocular nonphagocytic APC subset might migrate from the anterior uveal tissues, it is more probable that immune responses in the draining lymph nodes are engendered by soluble Ag escaping the eye through interstitial spaces. The inability of anterior uveal tissue APCs to migrate to lymph nodes may contribute to deviant immune responses that dominate after Ags are introduced into the anterior chamber.
Recruitment of Transferrin Receptor to Immunological Synapse in Response to TCR Engagement
Authors: Batista Alicia , Millán Jaime , Mittelbrunn María , Sánchez-Madrid Francisco , Alonso Miguel A. 
Published: April 2014

Abstract
T cell receptor engagement by an APC induces the formation of a highly organized complex of surface receptors and intracellular signaling molecules, known as the immunological synapse, at the site of cell-cell contact. The transferrin receptor (TfR, CD71) is normally present in the plasma membrane and recycling endosomes. In this study, we show that, although the TfR is typically absent from lipid rafts at steady state, stimulation with a mitogenic mixture of anti-CD3 Abs of human Jurkat T cells leads to a rapid compartmentalization of the TfR into lipid rafts accompanying that of CD3ε and activated Lck. This change occurs very rapidly and is accompanied by an increase in the surface expression of the TfR, probably by translocation from an internal endosomal pool. TfR recruitment to lipid rafts was also observed in primary T cells treated with mitogenic anti-CD3 Abs and in Jurkat T cell-APC conjugates. The use of beads coated with Abs indicates that the surface and endosomal TfR pools redistribute to the contact site region in response to engagement of CD28 and CD3. In T cell-APC conjugates, the T cell TfR endosomal pool relocates beneath the contact site, whereas surface TfR localizes to the peripheral ring of the immunological synapse. In the presence of specific anti-TfR Abs, the total number of T cell-APC contacts and the percentage of conjugates with CD3 and Lck translocated to the contact site were reduced. Our results therefore suggest the involvement of the TfR in the formation of the immunological synapse.
Unusual Chemokine Receptor Antagonism Involving a Mitogen-Activated Protein Kinase Pathway
Authors: Ogilvie Patricia , Thelen Sylvia , Moepps Barbara , Gierschik Peter , da Silva Campos Ana Claudia , Baggiolini Marco , Thelen Marcus 
Published: April 2014

Abstract
Antagonism of chemokines on chemokine receptors constitutes a new regulatory principle in inflammation. Eotaxin (CCL11), an agonist for CCR3 and an attractant of eosinophils, basophils, and Th2 lymphocytes, was shown to act as an antagonist for CCR2, which is widely expressed on leukocytes and is essential for inflammatory responses. In this report we provide direct evidence for a novel mechanism how chemokine receptor function can be arrested by endogenous ligands. We show that binding of eotaxin to CCR2 stimulates the mitogen-activated protein kinases extracellular signal-regulated kinase 1/2 (ERK1/2). Activation of the mitogen-activated protein kinase kinase 1/2-ERK pathway is indispensable for eotaxin-mediated attenuation of CCR2 function, as inhibition of ERK phosphorylation abolishes the arresting effect. ERK is also activated by CCR2 agonists, e.g., monocyte chemoattractant protein-1 (CCL2). However, the involved pathways are different, although in either case coupling of CCR2 to pertussis toxin-sensitive heterotrimeric G proteins is necessary. The results are in agreement with the view that CCR2 could assume different activation states depending on the ligand it encounters. With respect to actin polymerization and calcium mobilization, the different activation states lead to agonistic and antagonistic responses. It is conceivable that the intracellular signal transduction pathway that is activated by eotaxin could cause an attenuation of proinflammatory responses mediated by CCR2.
A Signal through OX40 (CD134) Allows Anergic, Autoreactive T Cells to Acquire Effector Cell Functions
Authors: Lathrop Stephanie K. , Huddleston Cortny A. , Dullforce Per A. , Montfort Megan J. , Weinberg Andrew D. , Parker David C. 
Published: April 2014

Abstract
To study mechanisms of peripheral self-tolerance, we injected small numbers of naive CD4+ TCR-transgenic T cells into mice expressing the MHC/peptide ligand under the control of an MHC class II promoter. The donor T cells expand rapidly to very large numbers, acquire memory markers, and go out into tissues, but the animals remain healthy, and the accumulated T cells are profoundly anergic to restimulation with Ag in vitro. Provision of a costimulatory signal by coinjection of an agonist Ab to OX40 (CD134), a TNFR family member expressed on activated CD4 T cells, results in death of the mice within 12 days. TCR-transgenic T cells recovered at 5 days from anti-OX40-treated mice have a unique phenotype: they remain unresponsive to Ag in vitro, but they are larger, more granular, and strongly IL-2R positive. Some spontaneously secrete IFN-γ directly ex vivo, and the majority make IFN-γ in response to PMA and ionomycin. Although they are anergic by conventional tests requiring Ag recognition, they respond vigorously to cytokines, proliferating in response to IL-2, and secreting IFN-γ when TCR signaling is bypassed with IL-12 and IL-18. We conclude that the costimulatory signal through OX40 allows otherwise harmless, proliferating, autoreactive T cells to acquire effector cell functions. The ability of these T cells to respond to cytokines by synthesizing additional inflammatory cytokines without a TCR signal may drive the fatal pathogenic process in vivo.
Methylation of STAT6 Modulates STAT6 Phosphorylation, Nuclear Translocation, and DNA-Binding Activity
Authors: Chen Weiguo , Daines Michael O. , Hershey Gurjit K. Khurana 
Published: April 2014

AbstractSignal transducer and activator of transcription 6 is a transcription factor important for the development of Th2 cells and regulation of gene expression by IL-4 and IL-13. It has been reported that STAT1 activity is regulated by methylation of a conserved arginine residue in the N-terminal domain. Methylation of STAT6 has not yet been explored. We observed methylation of STAT6 in cells transfected with wild-type STAT6, but not in cells transfected with Arg27Ala mutant, confirming that STAT6 is methylated on Arg27. Transfectants expressing mutant Arg27Ala STAT6 displayed markedly diminished IL-4-dependent STAT6 phosphorylation and nuclear translocation, and no STAT6 DNA-binding activity compared with wild-type STAT6 transfectants. To confirm this, the experiments were repeated using inhibitors of methylation. In the presence of methylation inhibitors, STAT6 methylation was diminished, as was phosphorylation of STAT6 and STAT6 DNA-binding activity. Thus, methylation is a critical regulator of STAT6 activity, necessary for optimal STAT6 phosphorylation, nuclear translocation, and DNA-binding activity. Furthermore, methylation of STAT6 has distinct effects from those reported with STAT1.
Pulmonary Surfactant Protein A Inhibits Macrophage Reactive Oxygen Intermediate Production in Response to Stimuli by Reducing NADPH Oxidase Activity
Authors: Crowther Joy E. , Kutala Vijay Kumar , Kuppusamy Periannan , Ferguson J. Scott , Beharka Alison A. , Zweier Jay L. , McCormack Francis X. , Schlesinger Larry S. 
Published: April 2014

Abstract
Alveolar macrophages are important host defense cells in the human lung that continuously phagocytose environmental and infectious particles that invade the alveolar space. Alveolar macrophages are prototypical alternatively activated macrophages, with up-regulated innate immune receptor expression, down-regulated costimulatory molecule expression, and limited production of reactive oxygen intermediates (ROI) in response to stimuli. Surfactant protein A (SP-A) is an abundant protein in pulmonary surfactant that has been shown to alter several macrophage (Mφ) immune functions. Data regarding SP-A effects on ROI production are contradictory, and lacking with regard to human Mφ. In this study, we examined the effects of SP-A on the oxidative response of human Mφ to particulate and soluble stimuli using fluorescent and biochemical assays, as well as electron paramagnetic resonance spectroscopy. SP-A significantly reduced Mφ superoxide production in response to the phorbol ester PMA and to serum-opsonized zymosan (OpZy), independent of any effect by SP-A on zymosan phagocytosis. SP-A was not found to scavenge superoxide. We measured Mφ oxygen consumption in response to stimuli using a new oxygen-sensitive electron paramagnetic resonance probe to determine the effects of SP-A on NADPH oxidase activity. SP-A significantly decreased Mφ oxygen consumption in response to PMA and OpZy. Additionally, SP-A reduced the association of NADPH oxidase component p47phox with OpZy phagosomes as determined by confocal microscopy, suggesting that SP-A inhibits NADPH oxidase activity by altering oxidase assembly on phagosomal membranes. These data support an anti-inflammatory role for SP-A in pulmonary homeostasis by inhibiting Mφ production of ROI through a reduction in NADPH oxidase activity.
The Upper and Lower Respiratory Tracts Differ in Their Requirement of IFN-γ and IL-4 in Controlling Respiratory Mycoplasma Infection and Disease
Authors: Woolard Matthew D. , Hodge Lisa M. , Jones Harlan P. , Schoeb Trenton R. , Simecka Jerry W. 
Published: April 2014

Abstract
The purpose of this study is to evaluate the significance of IFN-γ and IL-4 production in controlling mycoplasma infection and the pathogenesis of disease in the upper and lower respiratory tract. By using IFN-γ knockout and IL-4 knockout BALB/c mice, we were able to study the contribution of these cytokines in the development of pathogenesis and/or protection in response to mycoplasma respiratory infection, in both the upper and lower respiratory tracts. The loss of either IFN-γ or IL-4 does not affect disease pathogenesis or mycoplasma organism numbers in the upper respiratory tract. However, in the absence of IL-4, the nasal passages developed a compensatory immune response, characterized by higher numbers of macrophages and CD8+ T cells, which may be masking detrimental effects due to IL-4 deficiency. This is in contrast to the lower respiratory tract, where the loss of IFN-γ, but not IL-4, leads to higher mycoplasma numbers and increased disease severity. The loss of IFN-γ impacted the innate immune system’s ability to effectively clear mycoplasma, as the number of organisms was higher by day 3 postinfection. This higher organism burden most likely impacted disease pathogenesis; however, the development of Th2 cell-mediated adaptive immune response most likely contributed to lesion severity at later time points during infection. Our studies demonstrate that the upper and lower respiratory tracts are separate and distinct in their cytokine requirements for generating immunity against mycoplasma infection.
Induction of In Vivo Functional Db-Restricted Cytolytic T Cell Activity against a Putative Phosphate Transport Receptor of Mycobacterium tuberculosis
Authors: Romano Marta , Denis Olivier , D’Souza Sushila , Wang Xiao-Ming , Ottenhoff Tom H. M. , Brulet Jean-Marc , Huygen Kris 
Published: April 2014

Abstract
Using plasmid vaccination with DNA encoding the putative phosphate transport receptor PstS-3 from Mycobacterium tuberculosis and 36 overlapping 20-mer peptides spanning the entire PstS-3 sequence, we determined the immunodominant Th1-type CD4+ T cell epitopes in C57BL/10 mice, as measured by spleen cell IL-2 and IFN-γ production. Furthermore, a potent IFN-γ-inducing, Db-restricted CD8+ epitope was identified using MHC class I mutant B6.C-H-2bm13 mice and intracellular IFN-γ and whole blood CD8+ T cell tetramer staining. Using adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA vaccinated or M. tuberculosis-infected recipients, we demonstrated a functional in vivo CTL activity against this Db-restricted PstS-3 epitope. IFN-γ ELISPOT responses to this epitope were also detected in tuberculosis-infected mice. The CD4+ and CD8+ T cell epitopes defined for PstS-3 were completely specific and not recognized in mice vaccinated with either PstS-1 or PstS-2 DNA. The H-2 haplotype exerted a strong influence on immune reactivity to the PstS-3 Ag, and mice of the H-2b, p, and f haplotype produced significant Ab and Th1-type cytokine levels, whereas mice of H-2d, k, r, s, and q haplotype were completely unreactive. Low responsiveness against PstS-3 in MHC class II mutant B6.C-H-2bm12 mice could be overcome by DNA vaccination. IFN-γ-producing CD8+ T cells could also be detected against the Db-restricted epitope in H-2p haplotype mice. These results highlight the potential of DNA vaccination for the induction and characterization of CD4+ and particularly CD8+ T cell responses against mycobacterial Ags.
Intracellular and Cell Surface Localization of a Complex between αMβ2 Integrin and Promatrix Metalloproteinase-9 Progelatinase in Neutrophils
Authors: Stefanidakis Michael , Ruohtula Terhi , Borregaard Niels , Gahmberg Carl G. , Koivunen Erkki 
Published: April 2014

Abstract
We have recently demonstrated that promatrix metalloproteinases (proMMPs), particularly proMMP-9, are potent ligands of the leukocyte β2 integrins. We studied here the complex formation between proMMP-9 and αMβ2, the major MMP and integrin of neutrophils. On resting neutrophils, the proMMP-9/αMβ2 complex was primarily detected in intracellular granules, but after cellular activation it became localized to the cell surface, as demonstrated by immunoprecipitation and double immunofluorescence. Further indication of the complex formation was that neutrophils and αMβ2-transfected L cells, but not the wild-type L cells or leukocyte adhesion deficiency cells, bound to immobilized proMMP-9 or its recombinant catalytic domain in a β2 integrin-dependent manner. Peptides that bound to the αM integrin-I domain and inhibited its complex formation with proMMP-9 prevented neutrophil migration in a transendothelial assay in vitro and in a thioglycolate-elicited peritonitis in vivo. These results suggest that the translocating proMMP-9/αMβ2 complex may be part of the cell surface machinery guiding neutrophil migration.