bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Activation of IFN Regulatory Factor (IRF)-3 and IRF-5 Transcription Factors during Viral Infection
Authors: Cheng Tsu-Fan , Brzostek Sabrina , Ando Osamu , Van Scoy Sarah , Kumar K. Prasanna , Reich Nancy C.
Published: April 2014

Members of the IFN regulatory factor (IRF) family regulate gene expression critical to immune response, hemopoiesis, and proliferation. Although related by homology at their N-terminal DNA-binding domain, they display individual functional properties. The distinct properties result from differences in regulated expression, response to activating signals, and interaction with DNA regulatory elements. IRF-3 is expressed ubiquitously and is activated by serine phosphorylation in response to viral infection or TLR signaling. Evidence indicates that the kinases TANK-binding kinase 1 and inhibitor of NF-κB kinase-ε specifically phosphorylate and thereby activate IRF-3. We evaluated the contribution of another member of the IRF family, IRF-5, during viral infection since prior studies provided varied results. Analysis of phosphorylation, nuclear translocation, dimerization, binding to CREB-binding protein, recognition of DNA, and induction of gene expression were used comparatively with IRF-3 as a measure of IRF-5 activation. IRF-5 was not activated by viral infection; however, expression of TANK-binding kinase 1 or inhibitor of NF-κB kinase-ε did provide clear activation of IRF-5. IRF-5 is therefore distinct in its activation profile from IRF-3. However, similar to the biological effects of IRF-3 activation, a constitutively active mutation of IRF-5 promoted apoptosis. The apoptosis was inhibited by expression of Bcl-xL but not a dominant-negative mutation of the Fas-associated death domain. These studies support the distinct activation profiles of IRF-3 in comparison to IRF-5, but reveal a potential shared biological effect.
Molecular and Genetic Basis for Strain-Dependent NK1.1 Alloreactivity of Mouse NK Cells
Authors: Carlyle James R. , Mesci Aruz , Ljutic Belma , Belanger Simon , Tai Lee-Hwa , Rousselle Etienne , Troke Angela D. , Proteau Marie-France , Makrigiannis Andrew P.
Published: April 2014

NK1.1 alloantigen expression can be used to define NK cells in certain mouse strains, such as B6 (NKR-P1C) and SJL (NKR-P1B). However, BALB/c NK cells do not react with the anti-NK1.1 mAb, PK136. To investigate the NK1.1− phenotype of BALB/c NK cells, we have undertaken NK1.1 epitope mapping and genomic analysis of the BALB/c Nkrp1 region. Bacterial artificial chromosome library analysis reveals that, unlike the Ly49 region, the Nkrp1-Ocil/Clr region displays limited genetic divergence between B6 and BALB/c mice. In fact, significant divergence is confined to the Nkrp1b and Nkrp1c genes. Strikingly, the B6 Nkrp1d gene appears to represent a divergent allele of the Nkrp1b gene in BALB/c mice and other strains. Importantly, BALB/c NK cells express abundant and functional Nkrp1 transcripts, and the BALB/c NKR-P1B receptor functionally binds Ocil/Clr-b ligand. However, the BALB/c NKR-P1B/C sequences differ from those of the known NK1.1 alloantigens, and epitope mapping demonstrates that directed mutation of a single amino acid in the NKR-P1BBALB protein confers NK1.1 reactivity. Thus, PK136 mAb recognizes, in part, a distal C-terminal epitope present in NKR-P1BSw/SJL and NKR-P1CB6, but absent in NKR-P1A/D/FB6 and NKR-P1B/CBALB. Allelic divergence of the Nkrp1b/c gene products and limited divergence of the BALB/c Nkrp1-Ocil/Clr region explain a longstanding confusion regarding the strain-specific NK1.1 alloantigen reactivity of mouse NK cells.
Bacillus anthracisToxins Inhibit Human Neutrophil NADPH Oxidase Activity
Authors: Crawford Matthew A. , Aylott Caroline V. , Bourdeau Raymond W. , Bokoch Gary M.
Published: April 2014

Bacillus anthracis, the causative agent of anthrax, is a Gram-positive, spore-forming bacterium. B. anthracis virulence is ascribed mainly to a secreted tripartite AB-type toxin composed of three proteins designated protective Ag (PA), lethal factor, and edema factor. PA assembles with the enzymatic portions of the toxin, the metalloprotease lethal factor, and/or the adenylate cyclase edema factor, to generate lethal toxin (LTx) and edema toxin (ETx), respectively. These toxins enter cells through the interaction of PA with specific cell surface receptors. The anthrax toxins act to suppress innate immune responses and, given the importance of human neutrophils in innate immunity, they are likely relevant targets of the anthrax toxin. We have investigated in detail the effects of B. anthracis toxin on superoxide production by primary human neutrophils. Both LTx and ETx exhibit distinct inhibitory effects on fMLP (and C5a) receptor-mediated superoxide production, but have no effect on PMA nonreceptor-dependent superoxide production. These inhibitory effects cannot be accounted for by induction of neutrophil death, or by changes in stimulatory receptor levels. Analysis of NADPH oxidase regulation using whole cell and cell-free systems suggests that the toxins do not exert direct effects on NADPH oxidase components, but rather act via their respective effects, inhibition of MAPK signaling (LTx), and elevation of intracellular cAMP (ETx), to inhibit upstream signaling components mediating NADPH oxidase assembly and/or activation. Our results demonstrate that anthrax toxins effectively suppress human neutrophil-mediated innate immunity by inhibiting their ability to generate superoxide for bacterial killing.
A Novel Functional T Cell Hybridoma Recognizes Macrophage Cell Death Induced by Bacteria: A Possible Role for Innate Lymphocytes in Bacterial Infection
Authors: Kubota Koichi
Published: April 2014

We have established a novel TCRαβ (TCRVβ6)+CD4−CD8− T cell hybridoma designated B6HO3. When the B6HO3 cells were cocultured with bacterial-infected J774 macrophage-like cells, IFN-γ production by B6HO3 cells was triggered through direct cell-cell contact with dying J774 cells infected with Listeria monocytogenes (LM), Shigella flexneri, or Salmonella typhimurium that expressed the type III secretion system, but not with intact J774 cells infected with heat-killed LM, nonhemolytic lysteriolysin O-deficient (Hly−) LM, plasmid-cured Shigella, or stationary-phase Salmonella. However, the triggering of B6HO3 cells for IFN-γ production involved neither dying hepatoma cells infected with LM nor dying J774 cells caused by gliotoxin treatment or freeze thawing. Cycloheximide and Abs to H-2Kd, H-2Dd, Iad, CD1d, TCRVβ6, and IL-12 did not inhibit the contact-dependent IFN-γ response, indicating that this IFN-γ response did not require de novo protein synthesis in bacterial-infected J774 cells and was TCR and IL-12 independent. Thus, in an as yet undefined way, B6HO3 hybridoma recognizes a specialized form of macrophage cell death resulting from bacterial infection and consequently produces IFN-γ. Moreover, contact-dependent interaction of minor subsets of splenic αβ T cells, including NKT cells with dying LM-infected J774 and bone marrow-derived macrophage (BMM) cells, proved to provide an IFN-γ-productive stimulus for these minor T cell populations, to which the parental T cell of the B6HO3 hybridoma appeared to belong. Unexpectedly, subsets of γδ T and NK cells similarly responded to dying LM-infected macrophage cells. These results propose that innate lymphocytes may possess a recognition system sensing macrophage cell “danger” resulting from bacterial infection.
CXC Chemokine Ligand 13 Plays a Role in Experimental Autoimmune Encephalomyelitis
Authors: Bagaeva Ludmila V. , Rao Praveen , Powers James M. , Segal Benjamin M.
Published: April 2014

Experimental autoimmune encephalomyelitis (EAE) is a Tcell-mediated autoimmune disease of the CNS that is widely used as an animal model of multiple sclerosis. In this study, we investigate the role of CXCL13, a chemokine involved in the development and organization of secondary lymphoid tissues, in the pathogenesis of EAE. We detected CXCL13 mRNA and protein in spinal cords of mice with EAE. CXCL13-deficient mice exhibited a mild, self-limited form of disease. CXCL13 appeared to be important for the establishment of chronic white matter lesions. Furthermore, adoptive transfer experiments with CXCL13-deficient hosts indicate that the chemokine plays a distinct role during the effector phase. Our findings raise the possibility that reagents that antagonize or inhibit CXCL13 might be useful for the treatment of neuroinflammatory diseases such as multiple sclerosis.
Antitumor Activity of IFN-λ in Murine Tumor Models
Authors: Sato Atsuko , Ohtsuki Mamitaro , Hata Megumi , Kobayashi Eiji , Murakami Takashi
Published: April 2014

IFN-λ 1, -λ 2 and -λ 3 have been discovered as the latest members of the class II cytokine family and shown to possess antiviral activity. Murine B16 melanoma and Colon26 cancer cells were transduced with mouse IFN-λ to determine whether IFN-λ possesses antitumor activity. Overexpression of IFN-λ induced cell surface MHC class I expression and Fas/CD95 Ag, induced significant caspase-3/7 activity, and increased p21Waf1/Cip1 and dephosphorylated Rb (Ser780) in B16 cells in vitro. IFN-λ expression in tumor cell lines markedly inhibited s.c. and metastatic tumor formation in vivo compared with mock transfections (p < 0.05). Moreover, IFN-λ expression induced lymphocytic infiltrates, and an Ab-mediated immune cell depletion assay showed that NK cells were critical to IFN-λ-mediated tumor growth inhibition. Hydrodynamic injection of IFN-λ cDNA successfully targeted liver metastatic foci of Colon26 cells, and moderately decreased the mortality of mice with tumors. IFN-λ overexpression in the liver increased NK/NKT cells and enhanced their tumor-killing activity, and suggested the activation of innate immune responses. Thus, IFN-λ induced both tumor apoptosis and NK cell-mediated immunological tumor destruction through innate immune responses. These findings suggested that local delivery of IFN-λ might prove a useful adjunctive strategy in the clinical treatment of human malignancies.
NK Cells and γδ+ T Cells Are Phenotypically and Functionally Defective due to Preferential Apoptosis in Patients with Atopic Dermatitis
Authors: Katsuta Michie , Takigawa Yukio , Kimishima Momoko , Inaoka Miyuki , Takahashi Ryo , Shiohara Tetsuo
Published: April 2014

Innate immune cells mediate a first line of defense against pathogens and determine the nature of subsequent acquired immune responses, mainly by producing profound amounts of cytokines. Given these diverse tasks, it is predictable that defective NK and γδ+ T cell responses could be the underlying mechanism for the immunological alterations observed in atopic dermatitis (AD). Indeed, the frequencies of circulating NK cells and γδ+ T cells were profoundly reduced in AD patients. They also displayed a defective ability to sustain TNF-α and IFN-γ, but not IL-4, production after in vitro stimulation, and the defect was restricted to innate immune cells. Surprisingly, on the depletion of CD14+ monocytes, this selective impairment of TNF-α and IFN-γ production was restored to levels comparable to that observed in controls. Release of IL-10 from monocytes was not a major mechanism of the NK and γδ+ T cell dysfunction. Apoptosis as revealed by annexin V binding, was preferentially observed in NK and γδ+ T cells from AD patients when stimulated in the presence of monocytes, and depletion of monocytes significantly protected these cells from apoptotic cell death. Preferential apoptosis of NK cells by activated monocytes in AD patients was cell-contact-dependent. These results indicate that, once NK and γδ+ T cells in AD patients are in immediate contact with activated monocytes, these cells are specifically targeted for apoptosis, leading to the reduced type 1 cytokine production, thereby directing subsequent acquired immune responses toward a type-2 pattern and increasing susceptibility to infection.
Bone Marrow Mesenchymal Stem Cells Suppress Lymphocyte Proliferation In Vitro but Fail to Prevent Graft-versus-Host Disease in Mice
Authors: Sudres Muriel , Norol Françoise , Trenado Aurélie , Grégoire Sylvie , Charlotte Frédéric , Levacher Béatrice , Lataillade Jean-Jacques , Bourin Philippe , Holy Xavier , Vernant Jean-Paul , Klatzmann David , Cohen José L.
Published: April 2014

Several reports have suggested that mesenchymal stem cells (MSCs) could exert a potent immunosuppressive effect in vitro, and thus may have a therapeutic potential for T cell-dependent pathologies. We aimed to establish whether MSCs could be used to control graft-vs-host disease (GVHD), a major cause of morbidity and mortality after allogeneic hemopoietic stem cell transplantation. From C57BL/6 and BALB/c mouse bone marrow cells, we purified and expanded MSCs characterized by the lack of expression of CD45 and CD11b molecules, their typical spindle-shaped morphology, together with their ability to differentiate into osteogenic, chondrogenic, and adipogenic cells. These MSCs suppressed alloantigen-induced T cell proliferation in vitro in a dose-dependent manner, independently of their MHC haplotype. However, when MSCs were added to a bone marrow transplant at a MSCs:T cells ratio that provided a strong inhibition of the allogeneic responses in vitro, they yielded no clinical benefit on the incidence or severity of GVHD. The absence of clinical effect was not due to MSC rejection because they still could be detected in grafted animals, but rather to an absence of suppressive effect on donor T cell division in vivo. Thus, in these murine models, experimental data do not support a significant immunosuppressive effect of MSCs in vivo for the treatment of GVHD.
Differential Effects of IL-15 and IL-21 in Myeloid (CD11b+) and Lymphoid (CD11b−) Bone Marrow Cells
Authors: Pelletier Martin , Girard Denis
Published: April 2014

IL-15 has been found to activate NF-κB in various types of cells. However, the role of this transcription factor in IL-15- and IL-21-stimulated murine bone marrow (BM) cells is unclear. In this study, we demonstrated that both IL-15 and IL-21 are capable of delaying BM cell factor deprivation-induced apoptosis, but only IL-15 induced their proliferation. Following separation of BM cells into myeloid (CD11b+) and lymphoid (CD11b−) cell populations, we found that IL-15, but not IL-21, significantly induced proliferation in both cell populations. Both cytokines significantly delayed apoptosis, but only in CD11b− BM cells. IL-15Rα, CD122 (IL-2/15Rβ), and common γ-chains (CD132) were expressed in both populations, whereas IL-21Rα was expressed only in CD11b− BM cells. In addition, we demonstrated that IL-15-induced BM cell proliferation was significantly inhibited in NF-κBp50−/− mice when compared with littermate controls. The ability of IL-15 and IL-21 to delay BM cell apoptosis was slightly inhibited in NF-κBp50−/− mice, whereas the antiapoptotic effect of LPS was markedly reversed. We conclude that IL-15, but not IL-21, induces BM cell proliferation and that both cytokines delay BM cell apoptosis. These biological activities were preferentially observed in CD11b− BM cells. Using NF-κBp50−/− mice, we demonstrated for the first time that NF-κB plays a greater role in IL-15-induced cell proliferation than in IL-15- and IL-21-induced suppression of apoptosis.
The Basic Helix-Loop-Helix Transcription Factor HEBAlt Is Expressed in Pro-T Cells and Enhances the Generation of T Cell Precursors
Authors: Wang Duncheng , Claus Carol L. , Vaccarelli Giovanna , Braunstein Marsela , Schmitt Thomas M. , Zúñiga-Pflücker Juan Carlos , Rothenberg Ellen V. , Anderson Michele K.
Published: April 2014

The basic helix-loop-helix (bHLH) transcription factors HEB and E2A are critical mediators of gene regulation during lymphocyte development. We have cloned a new transcription factor, called HEBAlt, from a pro-T cell cDNA library. HEBAlt is generated by alternative transcriptional initiation and splicing from the HEB gene locus, which also encodes the previously characterized E box protein HEBCan. HEBAlt contains a unique N-terminal coding exon (the Alt domain) that replaces the first transactivation domain of HEBCan. Downstream of the Alt domain, HEBAlt is identical to HEBCan, including the DNA binding domain. HEBAlt is induced in early thymocyte precursors and down-regulated permanently at the double negative to double positive (DP) transition, whereas HEBCan mRNA expression peaks at the DP stage of thymocyte development. HEBAlt mRNA is up-regulated synergistically by a combination of HEBCan activity and Delta-Notch signaling. Retroviral transduction of HEBAlt or HEBCan into hemopoietic stem cells followed by OP9-DL1 coculture revealed that HEBAlt-transduced precursors generated more early T lineage precursors and more DP pre-T cells than control transduced cells. By contrast, HEBCan-transduced cells that maintained high level expression of the HEBCan transgene were inhibited in expansion and progression through T cell development. HEB−/− fetal liver precursors transduced with HEBAlt were rescued from delayed T cell specification, but HEBCan-transduced HEB−/− precursors were not. Therefore, HEBAlt and HEBCan are functionally distinct transcription factors, and HEBAlt is specifically required for the efficient generation of early T cell precursors.
G Protein-Coupled Receptor 83 Overexpression in Naive CD4+CD25− T Cells Leads to the Induction of Foxp3+ Regulatory T Cells In Vivo
Authors: Hansen Wiebke , Loser Karin , Westendorf Astrid M. , Bruder Dunja , Pfoertner Susanne , Siewert Christiane , Huehn Jochen , Beissert Stefan , Buer Jan
Published: April 2014

Foxp3 functions as a lineage specification factor for the development of naturally occurring thymus-derived CD4+CD25+ regulatory T (Treg) cells. Recent evidence suggests that naive Foxp3−CD4+CD25− T cells can be converted in the periphery into Foxp3+ Treg cells. In this study, we have identified the G protein-coupled receptor (GPR)83 to be selectively up-regulated by CD4+CD25+ Treg cells of both murine and human origin in contrast to naive CD4+CD25− or recently activated T cells. Furthermore, GPR83 was induced upon overexpression of Foxp3 in naive CD4+CD25− T cells. Transduction of naive CD4+CD25− T cells with GPR83-encoding retroviruses did not confer in vitro suppressive activity. Nevertheless, GPR83-transduced T cells were able to inhibit the effector phase of a severe contact hypersensitivity reaction of the skin, indicating that GPR83 itself or GPR83-mediated signals conferred suppressive activity to conventional CD4+ T cells in vivo. Most strikingly, this in vivo acquisition of suppressive activity was associated with the induction of Foxp3 expression in GPR83-transduced CD4+ T cells under inflammatory conditions. Our results suggest that GPR83 might be critically involved in the peripheral generation of Foxp3+ Treg cells in vivo.
Follicular Dendritic Cells Are Related to Bone Marrow Stromal Cell Progenitors and to Myofibroblasts
Authors: Muñoz-Fernández Raquel , Blanco Francisco Javier , Frecha Cecilia , Martín Francisco , Kimatrai Maria , Abadía-Molina Ana Clara , García-Pacheco José Marcos , Olivares Enrique G.
Published: April 2014

Follicular dendritic cells (FDC) are involved in the presentation of native Ags to B cells during the secondary immune response. Some authors consider FDC to be hemopoietic cells, whereas others believe them to be mesenchymal cells. The low proportion of FDC in the lymphoid follicle, together with technical difficulties in their isolation, make these cells difficult to study. We show that Fibroblast Medium can be used successfully to isolate and maintain FDC lines. In this culture medium, we obtained 18 FDC lines from human tonsils, which proliferated for as long as 18 wk and showed a stable Ag phenotype as detected by flow cytometry and RT-PCR. FDC lines were CD45-negative and expressed Ags associated to FDC (CD21, CD23, CD35, CD40, CD73, BAFF, ICAM-1, and VCAM-1) and Ags specific for FDC (DRC-1, CNA.42, and HJ2). These cell lines were also able to bind B cells and secrete CXCL13, functional activities characteristic of FDC. Nevertheless, the additional expression of STRO-1, together with CD10, CD13, CD29, CD34, CD63, CD73, CD90, ICAM-1, VCAM-1, HLA-DR, alkaline phosphatase, and α-smooth muscle actin (α-SM actin) indicated that FDC are closely related to bone marrow stromal cell progenitors. The expression of α-SM actin also relates FDC with myofibroblasts. Like myofibroblasts, FDC lines expressed stress fibers containing α-SM actin and were able to contract collagen gels under the effect of TGFβ1 and platelet-derived growth factor. These findings suggest that FDC are a specialized form of myofibroblast and derive from bone marrow stromal cell progenitors.
TLR Activation of Langerhans Cell-Like Dendritic Cells Triggers an Antiviral Immune Response
Authors: Renn Claudia N. , Sanchez David Jesse , Ochoa Maria Teresa , Legaspi Annaliza J. , Oh Chang-Keun , Liu Philip T. , Krutzik Stephan R. , Sieling Peter A. , Cheng Genhong , Modlin Robert L.
Published: April 2014

Langerhans cells (LC) are a unique subset of dendritic cells (DC), present in the epidermis and serving as the first line of defense against pathogens invading the skin. To investigate the role of human LCs in innate immune responses, we examined TLR expression and function of LC-like DCs derived from CD34+ progenitor cells and compared them to DCs derived from peripheral blood monocytes (monocyte-derived DC; Mo-DC). LC-like DCs and Mo-DCs expressed TLR1–10 mRNAs at comparable levels. Although many of the TLR-induced cytokine patterns were similar between the two cell types, stimulation with the TLR3 agonist poly(I:C) triggered significantly higher amounts of the IFN-inducible chemokines CXCL9 (monokine induced by IFN-γ) and CXCL11 (IFN-γ-inducible T cell α chemoattractant) in LC-like DCs as compared with Mo-DCs. Supernatants from TLR3-activated LC-like DCs reduced intracellular replication of vesicular stomatitis virus in a type I IFN-dependent manner. Finally, CXCL9 colocalized with LCs in skin biopsy specimens from viral infections. Together, our data suggest that LCs exhibit a direct antiviral activity that is dependent on type I IFN as part of the innate immune system.
IL-2 In Vivo Activities and Antitumor Efficacy Enhanced by an Anti-IL-2 mAb
Authors: Kamimura Daisuke , Sawa Yukihisa , Sato Masae , Agung Eviryanti , Hirano Toshio , Murakami Masaaki
Published: April 2014

IL-2 is a potent immunostimulant and has been tested for clinical use, including in immunotherapy for cancers and HIV infection. Here we show that a widely used neutralizing anti-murine IL-2 mAb (S4B6) exhibits unexpected activities that enhance the treatment effects of IL-2 in vivo. Coinjection of the anti-IL-2 mAb with a plasmid carrying murine IL-2 cDNA significantly increased the serum IL-2 levels and induced a substantial increase in the division of CD8+ T and NK1.1high cells in vivo. Injection of the mAb premixed with recombinant murine IL-2 showed the same enhanced effect. A 5-day treatment with the anti-IL-2 mAb alone gradually increased the CD44highCD8+ population, and the increased population was maintained for >300 days, suggesting that the mAb can gradually maintain and potentially enhance the bioactivity of endogenous IL-2 for extended periods. Furthermore, combined treatment with the anti-IL-2 mAb plus the IL-2 plasmid markedly enhanced Ag-specific CTL activity in vivo and partially protected mice from tumor metastasis to the lungs, compared with the anti-IL-2 mAb or IL-2 plasmid alone. These results demonstrated IL-2-enhancing effects of the anti-IL-2 mAb in vivo and suggest that combining a neutralizing anti-IL-2 Ab with IL-2 gene delivery might be used effectively to enhance IL-2 functions in clinical applications.
Analysis of TLR4 Polymorphic Variants: New Insights into TLR4/MD-2/CD14 Stoichiometry, Structure, and Signaling
Authors: Rallabhandi Prasad , Bell Jessica , Boukhvalova Marina S. , Medvedev Andrei , Lorenz Eva , Arditi Moshe , Hemming Val G. , Blanco Jorge C. G. , Segal David M. , Vogel Stefanie N.
Published: April 2014

TLR4 is the signal-transducing receptor for structurally diverse microbial molecules such as bacterial LPS, respiratory syncytial virus fusion (F) protein, and chlamydial heat shock protein 60. Previous studies associated two polymorphic mutations in the extracellular domain of TLR4 (Asp299Gly and Thr399Ile) with decreased LPS responsiveness. To analyze the molecular basis for diminished responsiveness, site-specific mutations (singly or coexpressed) were introduced into untagged and epitope (Flag)-tagged wild-type (WT) TLR4 expression vectors to permit a direct comparison of WT and mutant signal transduction. Coexpression of WT TLR4, CD14, and MD-2 expression vectors in HEK293T cells was first optimized to achieve optimal LPS-induced NF-κB reporter gene expression. Surprisingly, transfection of cells with MD-2 at high input levels often used in the literature suppressed LPS-induced signaling, whereas supraoptimal CD14 levels did not. Under conditions where WT and polymorphic variants were comparably expressed, significant differences in NF-κB activation were observed in response to LPS and two structurally unrelated TLR4 agonists, chlamydial heat shock protein 60 and RSV F protein, with the double, cosegregating mutant TLR4 exhibiting the greatest deficiency. Overexpression of Flag-tagged WT and mutant vectors at input levels resulting in agonist-independent signaling led to equivalent NF-κB signaling, suggesting that these mutations in TLR4 affect appropriate interaction with agonist or coreceptor. These data provide new insights into the importance of stoichiometry among the components of the TLR4/MD-2/CD14 complex. A structural model that accounts for the diminished responsiveness of mutant TLR4 polymorphisms to structurally unrelated TLR4 agonists is proposed.
Structural Phylogenetic Analysis of Activation-Induced Deaminase Function
Authors: Ichikawa H. Travis , Sowden Mark P. , Torelli Andrew T. , Bachl Jürgen , Huang Pinwei , Dance Geoffrey S. C. , Marr Shauna H. , Robert Jacques , Wedekind Joseph E. , Smith Harold C. , Bottaro Andrea
Published: April 2014

In mammals, activation-induced deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) of Ig genes. SHM and CSR activities require separate regions within AID. A chromosome region maintenance 1 (CRM1)-dependent nuclear export signal (NES) at the AID C terminus is necessary for CSR, and has been suggested to associate with CSR-specific cofactors. CSR appeared late in AID evolution, during the emergence of land vertebrates from bony fish, which only display SHM. Here, we show that AID from African clawed frog (Xenopus laevis), but not pufferfish (Takifugu rubripes), can induce CSR in AID-deficient mouse B cells, although both are catalytically active in bacteria and mammalian cell systems, albeit at decreased level. Like mammalian AID, Takifugu AID is actively exported from the cell nucleus by CRM1, and the Takifugu NES can substitute for the equivalent region in human AID, indicating that all the CSR-essential NES motif functions evolutionarily predated CSR activity. We also show that fusion of the Takifugu AID catalytic domain to the entire human noncatalytic domain restores activity in mammalian cells, suggesting that AID features mapping within the noncatalytic domain, but outside the NES, influence its function.
B Cells and Dendritic Cells from Vκ8 Light Chain Transgenic Mice Activate MRL-lpr/gldCD4+ T Cells
Authors: Beaudette-Zlatanova Britte C. , Ling Tina , Shlomchik Mark J. , Marshak-Rothstein Ann , Rifkin Ian R.
Published: April 2014

Autoreactive CD4+ T cells are required for full expression of disease in human systemic lupus erythematosus and in spontaneous murine lupus. However, the Ag specificity of these CD4+ T cells remains largely unknown. Rheumatoid factor (RF) B cells function as highly efficient APCs by taking up immune complexes (IC) and presenting IC constituents to T cells. We hypothesized that Ag-specific CD4+ T cells in lupus-prone mice could be identified by stimulating the CD4+ T cells with RF B cells from AM14 RF BCR transgenic mice pulsed with IC containing lupus-associated autoantibodies and autoantigens. This approach identified several independent T cell lines that proliferated robustly in response to IC-pulsed spleen cells from the AM14 RF BCR transgenic mice. However, these T cells did not recognize an IC constituent. Instead, these T cells recognized a determinant dependent on the inheritance of the transgene-encoded Vκ8 L chain, most likely a neoantigen created by the insertion of the transgene into the genome. Additionally, although the precise nature of the neoantigen is not known, the T cells described in this report may provide a useful tool for examining the role of T cells in the RF autoantibody response.
Persistence and Function of Central and Effector Memory CD4+ T Cells following Infection with a Gastrointestinal Helminth
Authors: Zaph Colby , Rook Kathryn A. , Goldschmidt Michael , Mohrs Markus , Scott Phillip , Artis David
Published: April 2014

Immunity in the gastrointestinal tract is important for resistance to many pathogens, but the memory T cells that mediate such immunity are poorly characterized. In this study, we show that following sterile cure of a primary infection with the gastrointestinal parasite Trichuris muris, memory CD4+ T cells persist in the draining mesenteric lymph node and protect mice against reinfection. The memory CD4+ T cells that developed were a heterogeneous population, consisting of both CD62Lhigh central memory T cells (TCM) and CD62Llow effector memory T cells (TEM) that were competent to produce the Th type 2 effector cytokine, IL-4. Unlike memory T cells that develop following exposure to several other pathogens, both CD4+ TCM and TEM populations persisted in the absence of chronic infection, and, critically, both populations were able to transfer protective immunity to naive recipients. CD62LhighCD4+ TCM were not apparent early after infection, but emerged following clearance of primary infection, suggesting that they may be derived from CD4+ TEM. Consistent with this theory, transfer of CD62LlowCD4+ TEM into naive recipients resulted in the development of a population of protective CD62LhighCD4+ TCM. Taken together, these studies show that distinct subsets of memory CD4+ T cells develop after infection with Trichuris, persist in the GALT, and mediate protective immunity to rechallenge.
Agonistic Monoclonal Antibody Against CD40 Receptor Decreases Lymphocyte Apoptosis and Improves Survival in Sepsis
Authors: Schwulst Steven J. , Grayson Mitchell H. , DiPasco Peter J. , Davis Christopher G. , Brahmbhatt Tejal S. , Ferguson Thomas A. , Hotchkiss Richard S.
Published: April 2014

Sepsis causes a marked apoptosis-induced depletion of lymphocytes. The degree of lymphocyte apoptosis during sepsis strongly correlates with survival. CD40, a member of the TNFR family, is expressed on APCs and has potent antiapoptotic activity. In this study we determined whether an agonistic Ab against CD40 could protect lymphocytes from sepsis-induced apoptosis. Secondly, we examined potential antiapoptotic mechanisms of the putative protection. Lastly, we aimed to determine whether anti-CD40 treatment could improve survival in sepsis. CD1 mice were made septic by the cecal ligation and puncture method and treated postoperatively with anti-CD40 Ab. Treatment with anti-CD40 completely abrogated sepsis-induced splenic B cell death and, surprisingly, decreased splenic and thymic T cell death as well (p < 0.001). To investigate the mechanism of protection of anti-CD40 therapy on T cells, CD40 receptor expression was examined. As anticipated, the CD40 receptor was constitutively expressed on B cells, but, unexpectedly, splenic and thymic T cells were found to express CD40 receptor during sepsis. Furthermore, CD4+CD8− T cells were the predominant subtype of T cells expressing CD40 receptor during sepsis. Additionally, the antiapoptotic protein Bcl-xL was found to be markedly increased in splenic B and T cells as well as in thymic T cells after treatment with anti-CD40 Ab (p < 0.0025). Lastly, mice that were made septic in a double injury model of sepsis had improved survival after treatment with anti-CD40 as compared with controls (p = 0.05). In conclusion, anti-CD40 treatment increases Bcl-xL, provides nearly complete protection against sepsis-induced lymphocyte apoptosis, and improves survival in sepsis.
IL-17 Plays an Important Role in the Development of Experimental Autoimmune Encephalomyelitis
Authors: Komiyama Yutaka , Nakae Susumu , Matsuki Taizo , Nambu Aya , Ishigame Harumichi , Kakuta Shigeru , Sudo Katsuko , Iwakura Yoichiro
Published: April 2014

IL-17 is a proinflammatory cytokine that activates T cells and other immune cells to produce a variety of cytokines, chemokines, and cell adhesion molecules. This cytokine is augmented in the sera and/or tissues of patients with contact dermatitis, asthma, and rheumatoid arthritis. We previously demonstrated that IL-17 is involved in the development of autoimmune arthritis and contact, delayed, and airway hypersensitivity in mice. As the expression of IL-17 is also augmented in multiple sclerosis, we examined the involvement of this cytokine in these diseases using IL-17−/− murine disease models. We found that the development of experimental autoimmune encephalomyelitis (EAE), the rodent model of multiple sclerosis, was significantly suppressed in IL-17−/− mice; these animals exhibited delayed onset, reduced maximum severity scores, ameliorated histological changes, and early recovery. T cell sensitization against myelin oligodendrocyte glycoprotein was reduced in IL-17−/− mice upon sensitization. The major producer of IL-17 upon treatment with myelin digodendrocyte glycopritein was CD4+ T cells rather than CD8+ T cells, and adoptive transfer of IL-17−/− CD4+ T cells inefficiently induced EAE in recipient mice. Notably, IL-17-producing T cells were increased in IFN-γ−/− cells, while IFN-γ-producing cells were increased in IL-17−/− cells, suggesting that IL-17 and IFN-γ mutually regulate IFN-γ and IL-17 production. These observations indicate that IL-17 rather than IFN-γ plays a crucial role in the development of EAE.
Head Kidney-Derived Macrophages of Common Carp (Cyprinus carpio L.) Show Plasticity and Functional Polarization upon Differential Stimulation
Authors: Joerink Maaike , Ribeiro Carla M. S. , Stet René J. M. , Hermsen Trudi , Savelkoul Huub F. J. , Wiegertjes Geert F.
Published: April 2014

Cells from the myeloid lineage are pluripotent. To investigate the potential of myeloid cell polarization in a primitive vertebrate species, we phenotypically and functionally characterized myeloid cells of common carp (Cyprinus carpio L.) during culture. Flow cytometric analysis, Ab labeling of cell surface markers, and light microscopy showed the presence of a major population of heterogeneous macrophages after culture. These head kidney-derived macrophages can be considered the fish equivalent of bone marrow-derived macrophages and show the ability to phagocytose, produce radicals, and polarize into innate activated or alternatively activated macrophages. Macrophage polarization was based on differential activity of inducible NO synthase and arginase for innate and alternative activation, respectively. Correspondingly, gene expression profiling after stimulation with LPS or cAMP showed differential expression for most of the immune genes presently described for carp. The recently described novel Ig-like transcript 1 (NILT1) and the CXCR1 and CXCR2 chemokine receptors were up-regulated after stimulation with cAMP, an inducer of alternative activation in carp macrophages. Up-regulation of NILT1 was also seen during the later phase of a Trypanosoma carassii infection, where macrophages are primarily alternatively activated. However, NILT1 could not be up-regulated during a Trypanoplasma borreli infection, a model for innate activation. Our data suggest that NILT1, CXCR1, and CXCR2 could be considered markers for alternatively activated macrophages in fish.
Gender Differences in Murine Airway Responsiveness and Lipopolysaccharide-Induced Inflammation
Authors: Card Jeffrey W. , Carey Michelle A. , Bradbury J. Alyce , DeGraff Laura M. , Morgan Daniel L. , Moorman Michael P. , Flake Gordon P. , Zeldin Darryl C.
Published: April 2014

The roles of gender and sex hormones in lung function and disease are complex and not completely understood. The present study examined the influence of gender on lung function and respiratory mechanics in naive mice and on acute airway inflammation and hyperresponsiveness induced by intratracheal LPS administration. Basal lung function characteristics did not differ between naive males and females, but males demonstrated significantly greater airway responsiveness than females following aerosolized methacholine challenge as evidenced by increased respiratory system resistance and elastance (p < 0.05). Following LPS administration, males developed more severe hypothermia and greater airway hyperresponsiveness than females (p < 0.05). Inflammatory indices including bronchoalveolar lavage fluid total cells, neutrophils, and TNF-α content were greater in males than in females 6 h following LPS administration (p < 0.05), whereas whole-lung TLR-4 protein levels did not differ among treatment groups, suggesting that differential expression of TLR-4 before or after LPS exposure did not underlie the observed inflammatory outcomes. Gonadectomy decreased airway inflammation in males but did not alter inflammation in females, whereas administration of exogenous testosterone to intact females increased their inflammatory responses to levels observed in intact males. LPS-induced airway hyperresponsiveness was also decreased in castrated males and was increased in females administered exogenous testosterone. Collectively, these data indicate that airway responsiveness in naive mice is influenced by gender, and that male mice have exaggerated airway inflammatory and functional responses to LPS compared with females. These gender differences are mediated, at least in part, by effects of androgens.
A Regulatory Effect of the Balance between TNF-α and IL-6 in the Granulomatous and Inflammatory Response to Rhodococcus aurantiacus Infection in Mice
Authors: Yimin  , Kohanawa Masashi
Published: April 2014

After i.v. inoculation with Rhodococcus aurantiacus, wild-type (WT) mice develop nonnecrotic, epithelioid granulomas. Because a high level of TNF-α is observed during the initial phase postinfection, we examined the extent to which TNF-α contributes to granulomatous inflammation using TNF-α gene-deficient (TNF-α−/−) mice. Despite a lack of R. aurantiacus proliferation, TNF-α−/− mice displayed high mortality rates within 5 days postinfection, as well as a high level of IL-6 in their spleens. Histological examination showed an absence of granuloma formation in TNF-α−/− mice. Pretreatment of TNF-α−/− mice with rTNF-α failed to restore this granuloma formation but accelerated bacterial removal and cellular recruitment. This rTNF-α administration also attenuated IL-6 production, resulting in increased survival rates of TNF-α−/− mice. Heat-killed R. aurantiacus induced in vitro enhanced mRNA expression and production of IL-6 in macrophages and DCs from TNF-α−/− mice when compared with WT controls, and treatment of TNF-α−/− mouse cells with rTNF-α decreased the IL-6 secretion. Moreover, anti-TNF-α or anti-IL-6 treatment increased IL-6 or TNF-α production by WT mouse cells, respectively. These data suggest that the production of TNF-α and IL-6 can be negatively regulated by each other. Administration of rIFN-γ to TNF-α−/− mice caused immature granulomas in livers, and treatment with both rTNF-α and rIFN-γ led to the formation of mature granulomas. Overall, TNF-α appears crucial for bacterial clearance, cellular recruitment, and granuloma formation. The balance between TNF-α and IL-6 during the early phase of infection controls the development of the inflammatory response to R. aurantiacus infection.
FcγRIIa, Not FcγRIIb, Is Constitutively and Functionally Expressed on Skin-Derived Human Mast Cells
Authors: Zhao Wei , Kepley Christopher L. , Morel Penelope A. , Okumoto Lawrence M. , Fukuoka Yoshihiro , Schwartz Lawrence B.
Published: April 2014

The expression of FcγR by human skin-derived mast cells of the MCTC type was determined in the current study. Expression of mRNA was analyzed with microarray gene chips and RT-PCR; protein by Western blotting and flow cytometry; function by release of β-hexosaminidase, PGD2, leukotriene C4 (LTC4), IL-5, IL-6, IL-13, GM-CSF, and TNF-α. FcγRIIa was consistently detected along with FcεRI at the mRNA and protein levels; FcγRIIc was sometimes detected only by RT-PCR; but FcγRIIb, FcγRI, and FcγRIII mRNA and protein were not detected. FcγRIIa-specific mAb caused skin MCTC cells to degranulate and secrete PGD2, LTC4, GM-CSF, IL-5, IL-6, IL-13, and TNF-α in a dose-dependent fashion. FcεRI-specific mAb caused similar amounts of each mediator to be released with the exception of LTC4, which was not released by this agonist. Simultaneous but independent cross-linking of FcεRI and FcγRIIa did not substantially alter mediator release above or below levels observed with each agent alone. Skin MCTC cells sensitized with dust-mite-specific IgE and IgG, when coaggregated by Der p2, exhibited enhanced degranulation compared with sensitization with either IgE or IgG alone. These results extend the known capabilities of human skin mast cells to respond to IgG as well as IgE-mediated signals.
Hierarchy of α Fetoprotein (AFP)-Specific T Cell Responses in Subjects with AFP-Positive Hepatocellular Cancer
Authors: Liu Yang , Daley Sean , Evdokimova Viktoria N. , Zdobinski David D. , Potter Douglas M. , Butterfield Lisa H.
Published: April 2014

We identified a series of immunodominant and subdominant epitopes from α fetoprotein (AFP), restricted by HLA-A*0201, which are recognized by the human T cell repertoire. The four immunodominant epitopes have been tested for immunogenicity in vivo, in HLA-A*0201+AFP+ advanced stage hepatocellular cancer (HCC) patients, and have activated and expanded AFP-specific IFN-γ-producing T cells in these patients, despite high serum levels of this self Ag. Here, we have examined the frequency, function, and avidity of the T cells specific for subdominant epitopes from AFP. We find that T cells specific for several of these epitopes are of similar or higher avidity than those specific for immunodominant epitopes. We then tested the peripheral blood of subjects ex vivo with different levels of serum AFP for the hierarchy of response to epitopes from this Ag and find that HCC patients have detectable frequencies of circulating IFN-γ-producing AFP-specific CD8+ T cells to both immunodominant and subdominant epitopes. We find the immunodominant and subdominant peptide-specific T cells to be differentially expanded with different modes of Ag presentation. Whereas spontaneous and AFP protein-stimulated responses show evidence for immunodominance, AdVhAFP-transduced dendritic cell-stimulated responses were broader and not skewed. Importantly, these data identify subdominant epitopes from AFP that can activate high-avidity T cells, and that can be detected and expanded in HCC subjects. These subdominant epitope-specific T cells can also recognize tumor cells and may be important therapeutically.
CD48 Is an Allergen and IL-3-Induced Activation Molecule on Eosinophils
Authors: Munitz Ariel , Bachelet Ido , Eliashar Ron , Khodoun Marat , Finkelman Fred D. , Rothenberg Marc E. , Levi-Schaffer Francesca
Published: April 2014

Eosinophils are involved in a variety of allergic, parasitic, malignant, and idiopathic disorders by releasing a variety of factors including specific granule proteins, lipid mediators, and proinflammatory and immunoregulatory cytokines and chemokines. In addition, they interact with various cell types in the inflamed tissue. Yet, the mechanism of eosinophil activation is still poorly understood. Recently, we described the expression and function of the CD2-subfamily of receptors and especially 2B4 on human eosinophils. In this study we focus on CD48, the high-affinity ligand of 2B4. CD48 is a GPI-anchored protein involved in cellular activation, costimulation, and adhesion, but has not been studied on eosinophils. We demonstrate that human eosinophils from atopic asthmatics display enhanced levels of CD48 expression and that IL-3 up-regulates CD48 expression. Furthermore, cross-linking CD48 on human eosinophils triggers release of eosinophil granule proteins. Assessment of CD48 expression in a murine model of experimental asthma revealed that CD48 is induced by allergen challenge and partially regulated by IL-3. Additionally, anti-IL-3 reduces CD48 expression and the degree of airway inflammation. Thus, CD48 is an IL-3-induced activating receptor on eosinophils, likely involved in promoting allergic inflammation.
Nucleic Acid-Sensing TLRs as Modifiers of Autoimmunity
Authors: Deane Jonathan A. , Bolland Silvia
Published: April 2014

The immune system requires precise regulation of activating and inhibitory signals so that it can mount effective responses against pathogens while ensuring tolerance to self-components. Some of the most potent activation signals are triggered by innate immune molecules, particularly those in the TLR family. Recent studies have shown that engagement of TLRs plays a significant role in both innate and adaptive immunity. This review focuses on the ways that TLR function might contribute to the etiology of lupus-like syndromes in the context of an autoimmune-prone environment. By considering the sources, localization, and expression of both nucleic acids and the molecules that bind them, we discuss several ways that innate immunity can play a role in the development of systemic autoimmunity.
Cutting Edge: CD4 Is the Receptor for the Tick Saliva Immunosuppressor, Salp15
Authors: Garg Renu , Juncadella Ignacio J. , Ramamoorthi Nandhini , Ashish  , Ananthanarayanan Shobana K. , Thomas Venetta , Rincón Mercedes , Krueger Joanna K. , Fikrig Erol , Yengo Christopher M. , Anguita Juan
Published: April 2014

Salp15 is an Ixodes scapularis salivary protein that inhibits CD4+ T cell activation through the repression of TCR ligation-triggered calcium fluxes and IL-2 production. We show in this study that Salp15 binds specifically to the CD4 coreceptor on mammalian host T cells. Salp15 specifically associates through its C-terminal residues with the outermost two extracellular domains of CD4. Upon binding to CD4, Salp15 inhibits the subsequent TCR ligation-induced T cell signaling at the earliest steps including tyrosine phosphorylation of the Src kinase Lck, downstream effector proteins, and lipid raft reorganization. These results provide a molecular basis to understanding the immunosuppressive activity of Salp15 and its specificity for CD4+ T cells.
Functional Dichotomy in CD40 Reciprocally Regulates Effector T Cell Functions
Authors: Murugaiyan Gopal , Agrawal Reena , Mishra Gyan C. , Mitra Debashis , Saha Bhaskar
Published: April 2014

Activation of T cells requires signals through Ag-specific TCR and costimulatory molecules such as CD40L. Although the use of defined tumor Ags for the induction of protective T cells met with limited success, the CD40-CD40L interaction that was proposed to induce antitumor T cells did not prevent tumor growth completely. Using a model for prostate tumor, a leading cause of tumor-induced mortality in men, we show that the failure is due to a novel functional dichotomy of CD40 whereby it self-limits its antitumor functions by inducing IL-10. IL-10 prevents the CD40-induced CTL and TNF-α and IL-12 production, Th1 skewing, and tumor regression. Priming mice with tumor lysate-pulsed IL-10-deficient dendritic cells (DCs) or wild-type DC plus anti-IL-10 Ab establishes antitumor memory T cells that can transfer the protection into syngenic nude mice. Infusion of Ag-pulsed IL-10-deficient but not wild-type DCs back into syngenic mice results in successful therapeutic autovaccination. Thus, we demonstrate the IL-10-sensitive antitumor T cell memory formulating a novel prophylactic and therapeutic principle.