bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms of Vδ1 γδ T Cell Activation by Microbial Components
Authors: Das Hiranmoy , Sugita Masahiko , Brenner Michael B.
Published: April 2014

There are two major subsets of γδ T cell in humans. Vγ2Vδ2 T cells predominate in the circulation and significantly expand in vivo during a variety of infectious diseases. Ags identified for the Vδ2 T cells are nonpeptide phosphate, amine, and aminobisphosphonate compounds. In contrast, Vδ1-encoded TCRs account for the vast majority of γδ T cells in tissues such as intestine and spleen. Some of these T cells recognize CD1c and MHC class I-related chain B molecules. These T cells are cytotoxic and use both perforin- and Fas-mediated cytotoxicity. A fundamental question is how these γδ T cells are activated during microbial exposure to carry out effector functions. In this study, we provide evidence for a mechanism by which Vδ1 γδ T cells are activated by inflammatory cytokines in the context of the Vδ1 TCR. Dendritic cells are necessary as accessory cells for microbial Ag-mediated Vδ1 γδ T cell activation. Cytokine (IL-12), adhesion (LFA3/CD2, LFA1/ICAM1) and costimulatory (MHC class I-related chain B molecule/NK-activating receptor G2D) molecules play a significant role along with Vδ1 TCR in this activation.
Activation-Induced Cell Death Limits Effector Function of CD4 Tumor-Specific T Cells
Authors: Saff Rebecca R. , Spanjaard Elena S. , Hohlbaum Andreas M. , Marshak-Rothstein Ann
Published: April 2014

A number of studies have documented a critical role for tumor-specific CD4+ cells in the augmentation of immunotherapeutic effector mechanisms. However, in the context of an extensive tumor burden, chronic stimulation of such CD4+ T cells often leads to the up-regulation of both Fas and Fas ligand, and coexpression of these molecules can potentially result in activation-induced cell death and the subsequent loss of effector activity. To evaluate the importance of T cell persistence in an experimental model of immunotherapy, we used DO11 Th1 cells from wild-type, Fas-deficient, and Fas ligand-deficient mice as effector populations specific for a model tumor Ag consisting of an OVA-derived transmembrane fusion protein. We found that the prolonged survival of Fas-deficient DO11 Th1 cells led to a more sustained tumor-specific response both in vitro and in vivo. Importantly, both Fas- and Fas ligand-deficient Th1 cells delayed tumor growth and cause regression of established tumors more effectively than wild-type Th1 cells, indicating that resistance to activation-induced cell death significantly enhances T cell effector activity.
Differential Requirements for Endosomal Reduction in the Presentation of Two H2-Ed-Restricted Epitopes from Influenza Hemagglutinin
Authors: Sinnathamby Gomathinayagam , Maric Maja , Cresswell Peter , Eisenlohr Laurence C.
Published: April 2014

We examined the role of reduction in the presentation of two H2-Ed-restricted epitopes (site 1 epitope (S1) and site 3 epitope (S3)) occupying distinct domains of the influenza hemagglutinin major subunit that contains four intrachain disulfide bonds and is connected to the virion by one interchain bond. S3 is situated within the stalk region that unfolds in response to mild acidification, and loads onto recycling H2-Ed in the early endosome, while S1, located in the structurally constrained globular domain, loads onto nascent H2-Ed in the late endosome. Predicting dependence upon reduction for either epitope seemed plausible but the results from several approaches were clear: presentation of S1 but not S3 is reduction dependent. Surprisingly, IFN-γ-inducible lysosomal thiol reductase (GILT), the only reductase thus far known to be involved in MHC class II-restricted processing, is not necessary for the generation of S1. However, GILT is necessary for presentation of either epitope when the virus is pretreated with a reducible cross-linker. The results suggest that unfolding of the Ag, perhaps a prerequisite for proteolytic processing in many cases, proceeds either spontaneously in the early endosome or via reduction in a later endosome. They further imply mechanisms for GILT-independent reduction in the late endosome, with GILT perhaps being reserved for more intractable Ags.
Enhanced T Cell Proliferation in Mice Lacking the p85β Subunit of Phosphoinositide 3-Kinase
Authors: Deane Jonathan A. , Trifilo Matthew J. , Yballe Claudine M. , Choi Sangdun , Lane Thomas E. , Fruman David A.
Published: April 2014

Phosphoinositide 3-kinase activation is important for lymphocyte proliferation and survival. Disrupting the gene that encodes the major phosphoinositide 3-kinase regulatory isoform p85α impairs B cell development and proliferation. However, T cell functions are intact in the absence of p85α. In this study, we test the hypothesis that the related isoform p85β is an essential regulatory subunit for T cell signaling. Unexpectedly, T cells lacking p85β showed a marked increase in proliferation and decreased death when stimulated with anti-CD3 plus IL-2. Both CD4+ and CD8+ T cells completed more cell divisions. Transcriptional profiling revealed reduced levels of caspase-6 mRNA in p85β-deficient T cells, which was paralleled by reduced caspase-6 enzyme activity. Increased T cell accumulation was also observed in vivo following infection of p85β-deficient mice with mouse hepatitis virus. Together, these results suggest a unique role for p85β in limiting T cell expansion.
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

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

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

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

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

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

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

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

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

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

Signal 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

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

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

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

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.
Iron Chelator Triggers Inflammatory Signals in Human Intestinal Epithelial Cells: Involvement of p38 and Extracellular Signal-Regulated Kinase Signaling Pathways
Authors: Choi Eun-Young , Kim Eun-Cheol , Oh Hyun-Mee , Kim Soonhag , Lee Hyun-Ju , Cho Eun-Young , Yoon Kwon-Ha , Kim Eun-A , Han Weon-Cheol , Choi Suck-Chei , Hwang Joo-Yeon , Park Chan , Oh Berm-Seok , Kim Youngyoul , Kimm Ku-Chan , Park Kie-In , Chung Hun-Taeg , Jun Chang-Duk
Published: April 2014

Competition for cellular iron (Fe) is a vital component of the interaction between host and pathogen. Most bacteria have an obligate requirement for Fe to sustain infection, growth, and survival in host. To obtain iron required for growth, many bacteria secrete iron chelators (siderophores). This study was undertaken to test whether a bacterial siderophore, deferoxamine (DFO), could trigger inflammatory signals in human intestinal epithelial cells as a single stimulus. Incubation of human intestinal epithelial HT-29 cells with DFO increased the expression of IL-8 mRNA, as well as the release of IL-8 protein. The signal transduction study revealed that both p38 and extracellular signal-regulated kinase-1/2 were significantly activated in response to DFO. Accordingly, the selective inhibitors for both kinases, either alone or in combination, completely abolished DFO-induced IL-8 secretion, indicating an importance of mitogen-activated protein kinases pathway. These proinflammatory effects of DFO were, in large part, mediated by activation of Na+/H+ exchangers, because selective blockade of Na+/H+ exchangers prevented the DFO-induced IL-8 production. Interestingly, however, DFO neither induced NF-κB activation by itself nor affected IL-1β- or TNF-α-mediated NF-κB activation, suggesting a NF-κB-independent mechanism in DFO-induced IL-8 production. Global gene expression profiling revealed that DFO significantly up-regulates inflammation-related genes including proinflammatory genes, and that many of those genes are down-modulated by the selective mitogen-activated protein kinase inhibitors. Collectively, these results demonstrate that, in addition to bacterial products or cell wall components, direct chelation of host Fe by infected bacteria may also contribute to the evocation of host inflammatory responses.
Humanin, a Newly Identified Neuroprotective Factor, Uses the G Protein-Coupled Formylpeptide Receptor-Like-1 as a Functional Receptor
Authors: Ying Guoguang , Iribarren Pablo , Zhou Ye , Gong Wanghua , Zhang Ning , Yu Zu-Xi , Le Yingying , Cui Youhong , Wang Ji Ming
Published: April 2014

Alzheimer’s disease (AD) is characterized by overproduction of β amyloid peptides in the brain with progressive loss of neuronal cells. The 42-aa form of the β amyloid peptide (Aβ42) is implied as a major causative factor, because it is toxic to neurons and elicits inflammatory responses in the brain by activating microglial cells. Despite the overproduction of Aβ42, AD brain tissue also generates protective factor(s) that may antagonize the neurodestructive effect of Aβ42. Humanin is a gene cloned from an apparently normal region of an AD brain and encodes a 24-aa peptide. Both secreted and synthetic Humanin peptides protect neuronal cells from damage by Aβ42, and the effect of Humanin may involve putative cellular receptor(s). To elucidate the molecular identity of such receptor(s), we examined the activity of synthetic Humanin on various cells and found that Humanin induced chemotaxis of mononuclear phagocytes by using a human G protein-coupled formylpeptide receptor-like-1 (FPRL1) and its murine counterpart FPR2. Coincidentally, FPRL1 and FPR2 are also functional receptors used by Aβ42 to chemoattract and activate phagocytic cells. Humanin reduced the aggregation and fibrillary formation by suppressing the effect of Aβ42 on mononuclear phagocytes. In neuroblast cells, Humanin and Aβ42 both activated FPRL1; however, only Aβ42 caused apoptotic death of the cells, and its cytopathic effect was blocked by Humanin. We conclude that Humanin shares human FPRL1 and mouse FPR2 with Aβ42 and suggest that Humanin may exert its neuroprotective effects by competitively inhibiting the access of FPRL1 to Aβ42.
Platelet-Activating Factor Receptor Develops Airway Hyperresponsiveness Independently of Airway Inflammation in a Murine Asthma Model
Authors: Ishii Satoshi , Nagase Takahide , Shindou Hideo , Takizawa Hajime , Ouchi Yasuyoshi , Shimizu Takao
Published: April 2014

Lipid mediators play an important role in modulating inflammatory responses. Platelet-activating factor (PAF) is a potent proinflammatory phospholipid with eosinophil chemotactic activity in vitro and in vivo. We show in this study that mice deficient in PAF receptor exhibited significantly reduced airway hyperresponsiveness to muscarinic cholinergic stimulation in an asthma model. However, PAF receptor-deficient mice developed an eosinophilic inflammatory response at a comparable level to that of wild-type mice. These results indicate an important role for PAF receptor, downstream of the eosinophilic inflammatory cascade, in regulating airway responsiveness after sensitization and aeroallergen challenge.
CD56+ Cells Induce Steroid Resistance in B Cells Exposed to IL-15
Authors: Xu Qing , Goleva Elena , Ou Liang-Shiou , Li Ling-Bo , Leung Donald Y. M.
Published: April 2014

Interleukin-2 can induce steroid resistance in T cells. IL-15 shares biological activities with IL-2, as both cytokines use IL-2Rγ for signal transduction. We therefore sought to determine whether IL-15 contributes to induction of PBMC corticosteroid resistance. Surprisingly, we found that incubation of unfractionated PBMC with IL-15 for 48 h resulted in the inhibition of glucocorticoid receptor (GCR) nuclear translocation in response to dexamethasone (DEX) treatment in CD19-positive B cells significantly greater than CD19-negative non-B cells (p < 0.01). However, pure B cells incubated with IL-15 responded normally with nuclear translocation of GCR in response to steroids, but failed to translocate GCR when they were grown in the presence of CD19− cells. Coculture of B cells with CD3+ (T cells), CD14+ (monocytes), or CD56+ (NK and NKT cells) in the presence of IL-15 revealed that only CD56+ cells contributed to the steroid insensitivity of B cells. IL-15 stimulation significantly increased production of IL-4 by CD56+ cells (p < 0.02). Treatment of purified B cells with combination IL-15/IL-4 resulted in abrogation of glucocorticoid receptor nuclear translocation and the inability of DEX to suppress cytokine production by B cells. In the presence of IL-4-neutralizing Ab, when B cells were cocultured with CD56+ cells and IL-15, the B cells were found to be steroid sensitive, i.e., DEX induced GCR nuclear translocation. This study demonstrates that B cells develop steroid resistance in the presence of CD56+ cells after IL-15 stimulation. Furthermore, IL-15 and IL-4 have the capacity to induce B cell insensitivity to steroids.
BCL-6 Mutations in Pulmonary Lymphoproliferative Disorders: Demonstration of an Aberrant Immunological Reaction in HIV-Related Lymphoid Interstitial Pneumonia
Authors: Kurosu Katsushi , Weiden Michael D. , Takiguchi Yuichi , Rom William N. , Yumoto Norio , Jaishree Jagirdar , Nakata Koh , Kasahara Yasunori , Tanabe Nobuhiro , Tatsumi Koichiro , Mikata Atsuo , Kuriyama Takayuki
Published: April 2014

We used a PCR and sequence procedure to analyze the Ig VH gene and the mutations in the 5′ regulatory regions of BCL-6 genes in pulmonary lymphoproliferative disorders (mucosa-associated lymphoid tissue (MALT) lymphoma, HIV-related, EBV-related, and virus-negative lymphocytic interstitial pneumonia (LIP)). Eight of 20 (40%) pulmonary MALT lymphoma and 10 of 20 LIP (5 of 5 (100%) HIV-related, 2 of 5 (40%) EBV-related, and 3 of 10 (30%) virus-negative LIP) cases showed BCL-6 gene mutations. Intraclonal heterogeneity of the BCL-6 mutations was observed only in pulmonary MALT lymphoma cases whose Ig VH genes also showed intraclonal heterogeneity. Ongoing BCL-6 mutations might reflect re-entry into a germinal center pathway to further mutations. BCL-6 mutations in pulmonary MALT lymphoma and HIV-negative LIP showed some features (high transition to transversion ratio, standard polarity, and RGYW/WRCY bias) of Ig VH gene hypermutation, leading to the view that pulmonary MALT lymphomas and HIV-negative LIP are under the influence of germinal center hypermutation mechanisms. Because BCL-6 mutations in HIV-related LIP cases did not demonstrate features of Ig VH gene hypermutation, immunological reactions in HIV-related LIP are the result of a process different from that found in HIV-negative pulmonary lymphoproliferative disorders.
Stroma Cell-Derived Factor 1α Mediates Desensitization of Human Neutrophil Respiratory Burst in Synovial Fluid from Rheumatoid Arthritic Patients
Authors: Lenoir Monique , Djerdjouri Bahia , Périanin Axel
Published: April 2014

Classical chemoattractants such as fMLP or the complement factor C5a use G protein (Gi)-coupled receptors to stimulate both chemotaxis and production of reactive oxygen species (respiratory burst, RB) by polymorphonuclear leukocytes (PMN). The chemokine stroma cell-derived factor 1α (SDF1α) and its Gi-coupled receptor, CXCR4, regulate leukocyte trafficking and recruitment to the synovial fluid of rheumatoid arthritic patients (RA-SF). However, the role of SDF1α in the RB is unknown and was studied in this work in vitro with healthy PMN in the absence and presence of RA-SF. In healthy PMN, SDF1α failed to stimulate the RB, even though the p38 mitogen-activated protein kinase was activated to a similar level as in fMLP-stimulated PMN. In contrast, the SDF1α-mediated calcium transients and activation of phosphatidylinositol 3-kinase/Akt were partially deficient, while p44/42 mitogen-activated protein kinases were not activated. SDF1α actually desensitized weakly the fMLP-mediated RB of healthy PMN. This cross-inhibitory effect was amplified in PMN treated with RA-SF, providing a protection against the exacerbation of RB induced by C5a or fMLP. This SDF1α beneficial effect, which was prevented by the CXCR4 antagonist AMD3100, was associated with impairment of C5a- and fMLP-mediated early signaling events. Thus, although SDF1α promotes leukocyte emigration into rheumatoid synovium, our data suggest it cross-desensitizes the production of oxidant by primed PMN, a property that may be beneficial in the context of arthritis.
Topical Glucocorticoid Therapy Directly Induces Up-Regulation of Functional CXCR4 on Primed T Lymphocytes in the Aqueous Humor of Patients with Uveitis
Authors: Curnow S. John , Wloka Kaska , Faint Jeff M. , Amft Nicole , Cheung C. M. Gemmy , Savant Vijay , Lord Janet , Akbar Arne N. , Buckley Christopher D. , Murray Philip I. , Salmon Mike
Published: April 2014

Overexpression of the constitutive chemokine receptor CXCR4 has been shown to contribute to the accumulation of leukocytes at sites of chronic inflammation. Glucocorticoids are widely used to treat inflammatory disorders such as uveitis to considerable effect, yet paradoxically have been reported to increase CXCR4 expression in vitro. We show here that ocular lymphocytes isolated from patients with uveitis who had been treated with topical glucocorticoids expressed highly elevated levels of CXCR4. The up-regulation of CXCR4 could be reproduced in vitro by culture of CD4+ T cells with aqueous humor (AqH), indicating a role for the ocular microenvironment rather than preferential recruitment of CXCR4+ cells. Untreated uveitis and noninflammatory AqH up-regulated CXCR4 to a limited extent; this was dependent on TGF-β2. However, the highest levels of CXCR4 both in vivo and in vitro were found in the glucocorticoid-treated patients. Glucocorticoids appeared to be directly responsible for the induction of CXCR4 in treated patients, as the glucocorticoid receptor antagonist RU38486 inhibited the in vitro up-regulation by AqH from these patients. Dexamethasone selectively up-regulated CXCR4 in vitro, but not any of a wide range of other chemokine receptors. CXCL12, the ligand for CXCR4, was present in AqH under noninflammatory conditions, but the levels were low in untreated uveitis and undetectable in treated uveitis AqH. The importance of these results for the treatment of HIV patients with glucocorticoids is discussed as well as a role for glucocorticoid-induced CXCR4 up-regulation and CXCL12 down-regulation in controlling the migration of lymphocyte populations, resulting in resolution of inflammation.
Inhibition of B Cell Death Causes the Development of an IgA Nephropathy in (New Zealand White × C57BL/6)F1-bcl-2 Transgenic Mice
Authors: Marquina Regina , Díez Miguel A. , López-Hoyos Marcos , Buelta Luis , Kuroki Aki , Kikuchi Shuichi , Villegas Juan , Pihlgren Maria , Siegrist Claire-Anne , Arias Manuel , Izui Shozo , Merino Jesús , Merino Ramón
Published: April 2014

Little is known about the pathogenic mechanisms of IgA nephropathy, despite being the most prevalent form of glomerulonephritis in humans. We report in this study that in (New Zealand White (NZW) × C57BL/6)F1 mice predisposed to autoimmune diseases, the expression of a human bcl-2 (hbcl-2) transgene in B cells promotes a CD4-dependent lupus-like syndrome characterized by IgG and IgA hypergammaglobulinemia, autoantibody production, and the development of a fatal glomerulonephritis. Histopathological analysis of glomerular lesions reveals that the glomerulonephritis observed in these animals resembles that of human IgA nephropathy. The overexpression of Bcl-2 in B cells selectively enhances systemic IgA immune responses to T-dependent Ags. Significantly, serum IgA purified from (NZW × C57BL/6)F1-hbcl-2 transgenic mice, but not from nontransgenic littermates, shows reduced levels of galactosylation and sialylation and an increased ability to deposit in the glomeruli, as observed in human patients with IgA nephropathy. Our results indicate that defects in the regulation of B lymphocyte survival associated with aberrant IgA glycosylation may be critically involved in the pathogenesis of IgA nephropathy, and that (NZW × C57BL/6)F1-hbcl-2 Tg mice provide a new experimental model for this form of glomerulonephritis.
A Promoter Haplotype of the Immunoreceptor Tyrosine-Based Inhibitory Motif-Bearing FcγRIIb Alters Receptor Expression and Associates with Autoimmunity. II. Differential Binding of GATA4 and Yin-Yang1 Transcription Factors and Correlated Receptor Expression and Function
Authors: Su Kaihong , Li Xiaoli , Edberg Jeffrey C. , Wu Jianming , Ferguson Polly , Kimberly Robert P.
Published: April 2014

The immunoreceptor tyrosine-based inhibitory motif-containing FcγRIIb modulates immune function on multiple cell types including B cells, monocytes/macrophages, and dendritic cells. The promoter for the human FCGR2B is polymorphic, and the less frequent 2B.4 promoter haplotype is associated with the autoimmune phenotype of systemic lupus erythematosus. In the present study, we demonstrate that the 2B.4 promoter haplotype of FCGR2B has increased binding capacity for GATA4 and Yin-Yang1 (YY1) transcription factors in both B lymphocytes and monocytes, and that overexpression of GATA4 or YY1 enhances the FCGR2B promoter activity. The 2B.4 haplotype leads to elevated expression of the endogenous receptor in heterozygous donors by ≈1.5-fold as assessed on EBV-transformed cells, primary B lymphocytes, and CD14+ monocytes. This increased expression accentuates the inhibitory effect of FcγRIIb on B cell Ag receptor signaling, measured by Ca2+ influx and cell viability in B cells. Our results indicate that transcription factors GATA4 and YY1 are involved in the regulation of FcγRIIb expression, and that the expression variants of FcγRIIb lead to altered cell signaling, which may contribute to autoimmune pathogenesis in humans.
Adenovirus CD40 Ligand Gene Therapy Counteracts Immune Escape Mechanisms in the Tumor Microenvironment
Authors: Loskog Angelica , Dzojic Helena , Vikman Sofia , Ninalga Christina , Essand Magnus , Korsgren Olle , Totterman Thomas H.
Published: April 2014

Tumors exhibit immune escape properties that promote their survival. These properties include modulation of Ag presentation, secretion of immunosuppressive factors, resistance to apoptosis, and induction of immune deviation, e.g., shifting from Th1- to Th2-type responses. These escape mechanisms have proven to hamper several immunotherapeutic strategies, and efforts need to be taken to revert this situation. We have studied the immunological effects of introducing CD40 ligand (CD40L), a potent dendritic cell activation molecule, into the tumor micromilieu by adenoviral gene transfer. For this purpose, a murine bladder cancer model (MB49) was used in C57BL/6 mice. The MB49 cells are known to induce IL-10 in the tumor environment. IL-10 potently inhibits the maturation of dendritic cells and thereby also the activation of CTLs. In this paper we show that CD40L immunogene therapy suppresses IL-10 and TGF-β production (2-fold decrease) and induces a typical Th1-type response in the tumor area (200-fold increase in IL-12 production). The antitumor responses obtained were MB49 cell specific, and the cytotoxicity of the stimulated CD8+ cells could be blocked by IL-10. Adenovirus CD40L therapy was capable of regressing small tumors (five of six animals were tumor free) and inhibiting the progression of larger tumors even in the presence of other escape mechanisms, such as apoptosis resistance. Furthermore, CD40L-transduced MB49 cells promoted the maturation of dendritic cells (2-fold increase in IL-12) independently of IL-10. Our results argue for using adenovirus CD40L gene transfer, alone or in combination with other modalities, for the treatment of Th2-dominated tumors.