bluuebunny/crossref_metadata_2025 huggingface dataset:
The Balance Between Donor T Cell Anergy and Suppression Versus Lethal Graft-Versus-Host Disease Is Determined by Host Conditioning
Authors: Gonzalez Mercedes , Quezada Sergio A. , Blazar Bruce R. , Panoskaltsis-Mortari Angela , Rudensky Alexander Y. , Noelle Randolph J.
Published: April 2014

Graft-vs-host disease (GVHD) remains the most life-threatening complication following the transfer of allogeneic bone marrow into immunocompromised hosts. Transferred alloreactive T cells respond in a complex manner. While massive T cell expansion is observed upon entry into an allogeneic environment, anergy, apoptosis, and repertoire selection are also observed. The study presented here shows that alloreactive T cell expansion and differentiation vs anergy and suppression are dramatically influenced by host conditioning. Using alloreactive CD4+ and CD8+ TCR transgenic (Tg) T cells, a novel GVHD model is presented that allows for the visualization of how alloreactive T cells behave when host conditioning is manipulated. Following the transfer of alloreactive CD4+ and CD8+ TCR Tg T cells into sublethally irradiated hosts, both Tg T cells populations expand, develop effector function, and cause GVHD. In contrast, when Tg T cells are transferred in non-irradiated hosts, expansion is observed, but there is no development of effector function or disease. Assessment of CD4+ Tg T cell function following transfer into non-irradiated hosts reveals that these CD4+ Tg cells are profoundly anergic and have acquired a regulatory function, as manifested in their ability to suppress the expansion of naive TCR Tg T cells in vitro and in vivo as well as the development of GVHD. These findings underscore the decisive effect of the inflammatory environment created by irradiation in determining the ultimate fate and function of alloreactive T cells in vivo
Allogeneic Neonatal Neuronal Retina Grafts Display Partial Immune Privilege in the Subcapsular Space of the Kidney
Authors: Ng Tat Fong , Osawa Hideya , Hori Junko , Young Michael J. , Streilein J. Wayne
Published: April 2014

Transplantation of immature retinal tissues may offer a solution for restoring sight to individuals afflicted with degenerative retinal diseases. Promising results have recently demonstrated that neonatal retinal grafts placed in the eye can survive, differentiate into photoreceptor cells, and respond to evoked electrical stimuli. These transplants, however, were performed in immunologically immature recipients. Since it is important to know whether neonatal neuronal retina (NNR) tissue is immunogenic in immune-competent recipients, and whether this tissue displays inherent immune privilege, we have examined the fate of such grafts placed in a non-immune-privileged site of adult recipient mice. We found that typical, photoreceptor-dominated rosettes formed in differentiating NNR grafts, and that these allografts survived beyond 12 days, whereas genetically identical skin grafts were rejected earlier. Class II MHC-bearing cells of recipient origin were observed along the edge of NNR allografts as early as day 5. Donor-specific delayed hypersensitivity was not detected at 12 days, but did emerge on day 20, coincident with rejection of NNR allografts. Lymph nodes, but not spleens, of mice bearing NNR grafts at 12 days contained regulatory lymphoid cells that suppressed delayed hypersensitivity in naive recipients. We conclude that NNR grafts accommodate and even differentiate in the non-immune-privileged space beneath the kidney capsule. Survival beneath the kidney capsule of NNR allografts, but not skin allografts, at 12 days and beyond implies that NNR tissue possesses inherent immune privilege. The vulnerability of these grafts to rejection by 20 days reveals this privilege to be partial and temporary.
CD19 Signaling Pathways Play a Major Role for Murine AIDS Induction and Progression
Authors: Knoetig Sonja M. , Torrey Ted A. , Naghashfar Zohreh , McCarty Tom , Morse Herbert C.
Published: April 2014

Infection of genetically susceptible mice with the LP-BM5 mixture of murine leukemia viruses including an etiologic defective virus (BM5def) causes an immunodeficiency syndrome called murine AIDS (MAIDS). The disease is characterized by interactions between B cells and CD4+ T cells resulting in polyclonal activation of both cell types. It is known that BM5def is expressed at highest levels in B cells and that B cells serve as viral APC. The CD19-CD21 complex and CD22 on the surface of B cells play critical roles as regulators of B cell responses to a variety of stimuli, influencing cell activation, differentiation, and survival. CD19 integrates positive signals induced by B cell receptor ligation by interacting with the protooncogene Vav, which leads to subsequent tyrosine phosphorylation of this molecule. In contrast, CD22 negatively regulates Vav phosphorylation. To analyze the role of CD19, CD21, Vav, and CD22 in MAIDS, we infected mice deficient in CD19, CD21 (CR2), Vav-1, or CD22 with LP-BM5 murine leukemia viruses. Infected CR2−/− mice developed MAIDS with a time course and severity indistinguishable from that of wild-type mice. In contrast, CD19 as well as Vav-1 deficiency restricted viral replication and suppressed the development of typical signs of MAIDS including splenomegaly, lymphadenopathy, and hypergammaglobulinemia. Finally, CD22 deficiency was found to accelerate MAIDS development. These results provide novel insights into the B cell signaling pathways required for normal induction and progression of MAIDS.
Competition Between MHC Class I Alleles for Cell Surface Expression Alters CTL Responses to Influenza A Virus
Authors: Tourdot Sophie , Gould Keith G.
Published: April 2014

Mammalian cells express up to six different MHC class I alleles, many of which differ in terms of their interaction with components of the Ag presentation pathway and level of cell surface expression. However, it is often assumed in Ag presentation studies that class I alleles function independently of each other. We have compared cell surface expression levels and function of MHC class I molecules in F1 hybrid mice with those in the homozygous parental strains. The level of cell surface expression of certain alleles in F1 mice differed significantly from 50% of that found on the same cell type in the corresponding parental strain, suggesting allele-specific competition for cell surface expression, and not expression solely according to gene dosage. The strongest effect was observed in H-2b × H-2k F1 mice, in which the H-2b class I molecules dominated over the H-2k class I molecules. The magnitude of H-2k-restricted CTL responses to influenza A virus infection was similar in the F1 hybrid and parental H-2k mice. However, in H-2k mice expressing a Kb transgene, cell surface levels of the endogenous class I molecules were down-regulated to a greater degree than in F1 hybrid mice, and H-2k-restricted CTL responses against influenza A virus were greatly reduced, although the CTL repertoire was apparently present. Therefore, certain MHC class I molecules compete with each other for cell surface expression, and the resulting low cell surface expression of specific alleles can lead to a severe reduction in the ability to generate a CTL response.
Differential Regulation of CD44 Expression by Lipopolysaccharide (LPS) and TNF-α in Human Monocytic Cells: Distinct Involvement of c-Jun N-Terminal Kinase in LPS-Induced CD44 Expression
Authors: Gee Katrina , Lim Wilfred , Ma Wei , Nandan Devki , Diaz-Mitoma Francisco , Kozlowski Maya , Kumar Ashok
Published: April 2014

Alterations in the regulation of CD44 expression play a critical role in modulating cell adhesion, migration, and inflammation. LPS, a bacterial cell wall component, regulates CD44 expression and may modulate CD44-mediated biological effects in monocytic cells during inflammation and immune responses. In this study, we show that in normal human monocytes, LPS and LPS-induced cytokines IL-10 and TNF-α enhance CD44 expression. To delineate the mechanism underlying LPS-induced CD44 expression, we investigated the role of the mitogen-activated protein kinases (MAPKs), p38, p42/44 extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) by using their specific inhibitors. We demonstrate the involvement, at least in part, of p38 MAPK in TNF-α-induced CD44 expression in both monocytes and promonocytic THP-1 cells. However, neither p38 nor p42/44 MAPKs were involved in IL-10-induced CD44 expression in monocytes. To further dissect the TNF-α and LPS-induced signaling pathways regulating CD44 expression independent of IL-10-mediated effects, we used IL-10 refractory THP-1 cells as a model system. Herein, we show that CD44 expression induced by the LPS-mediated pathway predominantly involved JNK activation. This conclusion was based on results derived by transfection of THP-1 cells with a dominant-negative mutant of stress-activated protein/extracellular signal-regulated kinase kinase 1, and by exposure of cells to JNK inhibitors dexamethasone and SP600125. All these treatments prevented CD44 induction in LPS-stimulated, but not in TNF-α-stimulated, THP-1 cells. Furthermore, we show that CD44 induction may involve JNK-dependent early growth response gene activation in LPS-stimulated monocytic cells. Taken together, these results suggest a predominant role of JNK in LPS-induced CD44 expression in monocytic cells.
Regulation of Virus-Induced IL-12 and IL-23 Expression in Human Macrophages
Authors: Pirhonen Jaana , Matikainen Sampsa , Julkunen Ilkka
Published: April 2014

IL-23 is a novel cytokine that promotes the proliferation of naive and memory T cells and stimulates their IFN-γ production. Besides functional similarities, IL-23 bears structural resemblance to IL-12. Biologically active IL-23 is a heterodimer whose p40 subunit is identical to IL-12p40 while its p19 subunit is distantly related to IL-12p35. In the present study we demonstrate that human monocyte-derived macrophages are able to produce IL-23 in response to virus infection. Sendai virus stimulates the expression of p19 and p40 mRNAs in macrophages. Furthermore, it enhances p35 mRNA expression and the production of IL-12. Influenza A virus, in contrast, fails to stimulate IL-12 or IL-23 expression in macrophages. IL-12 and IL-23 contribute to the IFN-γ-inducing activity that cell culture supernatant from Sendai virus-infected macrophages show in NK-92 cells. The induction of IFN-γ production occurs in concert with IFN-αβ and IL-18, which are also secreted from the virus-infected cells. The IFN-γ-inducing activity is inhibited by IL-4, which down-regulates the transcription of p19 and p40 genes and the secretion of IFN-αβ, IL-12, and IL-18. IFN-γ, in contrast, up-regulates the p19 and p40 mRNA expression in Sendai virus infection. Thus, IL-4 and IFN-γ serve as opposing factors in the regulation of IFN-γ-inducing cytokines, including IL-23, in macrophages.
TGF-β1 Regulates Adhesion of Mucosal Mast Cell Homologues to Laminin-1 Through Expression of Integrin α7
Authors: Rosbottom Anne , Scudamore Cheryl L. , von der Mark Helga , Thornton Elizabeth M. , Wright Steven H. , Miller Hugh R. P.
Published: April 2014

Mucosal mast cells (MMC) or their precursors migrate through the intestinal lamina propria to reside intraepithelially, where expression of mouse mast cell protease-1 indicates the mature phenotype. Alterations in expression of integrins that govern cell adhesion to the extracellular matrix may regulate this process. As the key cytokine mediating differentiation of mouse mast cell protease-1-expressing MMC homologues in vitro, TGF-β1 was considered a likely candidate for regulation of the integrins that facilitate intraepithelial migration of MMC. Therefore, we examined adhesion of bone marrow-derived mast cells cultured with and without TGF-β1 to laminin-1, fibronectin, and vitronectin along with expression of integrins likely to regulate this adhesion. Adhesion of PMA-stimulated cultured mast cells to laminin-1 increased from 5.3 ± 3.6% (mean ± SEM) in the absence of TGF-β1 to 58.7 ± 4.0% (p < 0.05) when cultured mast cells had differentiated into MMC homologues in the presence of TGF-β1. Increased adhesion of MMC homologues to laminin-1 was also stimulated by FcεRI cross-linking and the calcium ionophore A23187. Expression of the laminin-binding integrin α7 by MMC homologues grown in the presence of TGF-β1 was demonstrated by RT-PCR and flow cytometry, and preincubation of MMC homologues with the α7-neutralizing Ab 6A11 inhibited adhesion to laminin-1 by 98% (p < 0.05), demonstrating a novel role for this molecule in adhesion of a hemopoietic cell to laminin-1.
Characterization of the Interaction Between L-Ficolin/P35 and Mannan-Binding Lectin-Associated Serine Proteases-1 and -2
Authors: Cseh Sandor , Vera Loanys , Matsushita Misao , Fujita Teizo , Arlaud Gérard J. , Thielens Nicole M.
Published: April 2014

Ficolins are oligomeric lectins comprising a collagen-like and a fibrinogen-like domain, with a binding specificity for N-acetylglucosamine. It has been reported recently that L-ficolin/P35 associates with mannan-binding lectin (MBL)-associated serine proteases (MASP-1 and -2) and MBL-associated protein 19 (MAp19) in serum and forms complexes able to activate complement. Using surface plasmon resonance spectroscopy we have shown that recombinant MASP-1 and -2, their N-terminal CUB1 (module originally found in complement proteins C1r/C1s, Uegf, and bone morphogenetic protein-1)-epidermal growth factor (EGF)-CUB2 and CUB1-EGF segments, and MAp19 bind to immobilized L-ficolin/P35 in the presence of Ca2+ ions. Comparable Kd values were obtained for the full-length proteases and their CUB1-EGF-CUB2 segments (9.2 and 10 nM for MASP-1 and 4.6 and 5.4 nM for MASP-2, respectively), whereas higher values were obtained for the CUB1-EGF segments (26.7, 15.6, and 14.3 nM for MASP-1, MASP-2, and MAp19). These values are in the same range as those determined for the interaction of these proteins with MBL. Binding was Ca2+ dependent and was only partly sensitive to EDTA for MASP-1, MASP-2, and MASP-2 CUB1-EGF-CUB2. Half-maximal binding was obtained at comparable Ca2+ concentrations for MASP-1 and MASP-2 (0.45 and 0.47 μM, respectively), their CUB1-EGF-CUB2 segments (0.37 and 0.72 μM), and their CUB1-EGF segments (0.31 and 0.79 μM). These values are lower than those determined in the case of MBL, indicating a difference between MBL and L-ficolin/P35 with respect to the Ca2+ dependence of their interaction with the MASPs. Preincubation of the MASPs with soluble MBL inhibited subsequent binding to immobilized L-ficolin/P35 and, conversely, suggesting that these lectins compete with each other for binding to the MASPs in vivo.
Proliferation Requirements of Cytomegalovirus-Specific, Effector-Type Human CD8+ T Cells
Authors: van Leeuwen Ester M. , Gamadia Laila E. , Baars Paul A. , Remmerswaal Ester B. , ten Berge Ineke J. , van Lier René A.
Published: April 2014

Two prototypic types of virus-specific CD8+ T cells can be found in latently infected individuals: CD45R0+CD27+CCR7− effector-memory, and CD45RA+CD27−CCR7− effector-type cells. It has recently been implied that CD45RA+CD27−CCR7− T cells are terminally differentiated effector cells and as such have lost all proliferative capacity. We show in this study, however, that stimulation of CMV-specific CD45RA+CD27−CCR7− T cells with their cognate peptide in concert with either CD4+ help or IL-2, IL-15, or IL-21 in fact induces massive clonal expansion. Concurrently, these stimulated effector T cells change cell surface phenotype from CD45RA to CD45R0 and regain CCR7, while effector functions are maintained. Our data imply that CD45RA+CD27−CCR7− effector-type T cells contribute to immunity not only by direct execution of effector functions, but also by yielding progeny in situations of viral reinfection or reactivation.
A Protease-Activated Pathway Underlying Th Cell Type 2 Activation and Allergic Lung Disease
Authors: Kheradmand Farrah , Kiss Attila , Xu Jie , Lee Seung-Hyo , Kolattukudy Pappachan E. , Corry David B.
Published: April 2014

The respiratory allergens that induce experimental Th cell type 2-dependent allergic lung inflammation may be grouped into two functional classes. One class of allergens, in this study termed type I, requires priming with adjuvants remote from the lung to overcome airway tolerogenic mechanisms that ordinarily preclude allergic responses to inhaled Ags. In contrast, the other, or type II, allergen class requires neither remote priming nor additional adjuvants to overcome airway tolerance and elicit robust allergic lung disease. In this study, we show in an experimental model that diverse type II allergens share in common proteolytic activity that is both necessary and sufficient for overcoming airway tolerance and induction of pulmonary allergic disease. Inactivated protease and protease-free Ag fragments showed no allergenic potency, demonstrating that only active protease acting on endogenous substrates was essential. Furthermore, induction of airway tolerance could be aborted and allergic lung disease established by simply adding purified protease to a type I allergen. Thus, exogenous proteases are common to type II allergens and may be generally required to overcome the innate resistance of the airway to Th cell type 2 activation and allergic inflammation, raising concern for their potential contribution to diseases such as asthma.
Absence of the Complement Anaphylatoxin C3a Receptor Suppresses Th2 Effector Functions in a Murine Model of Pulmonary Allergy
Authors: Drouin Scott M. , Corry David B. , Hollman Travis J. , Kildsgaard Jens , Wetsel Rick A.
Published: April 2014

Asthma is a chronic inflammatory disease of the lung resulting in airway obstruction. The airway inflammation of asthma is strongly linked to Th2 lymphocytes and their cytokines, particularly IL-4, IL-5, and IL-13, which regulate airway hyperresponsiveness, eosinophil activation, mucus production, and IgE secretion. Historically, complement was not thought to contribute to the pathogenesis of asthma. However, our previous reports have demonstrated that complement contributes to bronchial hyperreactivity, recruitment of airway eosinophils, IL-4 production, and IgE responses in a mouse model of pulmonary allergy. To define the complement activation fragments that mediate these effects, we assessed the role of the complement anaphylatoxin C3a in a mouse model of pulmonary allergy by challenging C3aR-deficient mice intranasally with a mixed Ag preparation of Aspergillus fumigatus cell culture filtrate and OVA. Analysis by plethysmography after challenge revealed an attenuation in airway hyperresponsiveness in C3aR-deficient mice relative to wild-type mice. C3aR-deficient mice also had an 88% decrease in airway eosinophils and a 59% reduction in lung IL-4-producing cells. Consistent with the reduced numbers of IL-4-producing cells, C3aR-deficient mice had diminished bronchoalveolar lavage levels of the Th2 cytokines, IL-5 and IL-13. C3aR knockout mice also exhibited decreases in IgE titers as well as reduced mucus production. Collectively, these data highlight the importance of complement activation, the C3a anaphylatoxin, and its receptor during Th2 development in this experimental model and implicate these molecules as possible therapeutic targets in diseases such as asthma.
Dysregulation of IFN-γ Signaling Pathways in the Absence of TGF-β1
Authors: McCartney-Francis Nancy L. , Wahl Sharon M.
Published: April 2014

Deficiency of TGF-β1 is associated with immune dysregulation and autoimmunity as exemplified by the multifocal inflammatory lesions and early demise of the TGF-β1 null mice. Elevated NO metabolites (nitrite and nitrate) in the plasma of these mice suggest a participatory role of NO in the pathogenic inflammatory response. To determine the mechanism for this dysregulation, we examined upstream elements that could contribute to the overexpression of NO, including inducible NO synthase (iNOS) and transcription factors Stat1α and IFN-regulatory factor-1 (IRF-1). The coincident up-regulation of IFN-γ, an iNOS inducer, and iNOS, before the appearance of inflammatory lesions, suggests that failed regulation of the IFN-γ signaling pathway may underlie the immunological disorder in TGF-β1 null mice. In fact, IFN-γ-driven transcription factors IRF-1 and Stat1α, both of which act as transcriptional activators of iNOS, were elevated in the null mice. Treatment of mice with a polyclonal anti-IFN-γ Ab reduced expression and activity not only of transcription factors Stat1α and IRF-1 but also of iNOS. Furthermore, anti-IFN-γ treatment delayed the cachexia normally seen in TGF-β1 null mice and increased their longevity. The global nature of immune dysregulation in TGF-β1 null mice documents TGF-β1 as an essential immunoregulatory molecule.
Expression and Function of C5a Receptor in Mouse Microvascular Endothelial Cells
Authors: Laudes Ines J. , Chu Jeffrey C. , Huber-Lang Markus , Guo Ren-Feng , Riedemann Niels C. , Sarma J. Vidya , Mahdi Fakhri , Murphy Hedwig S. , Speyer Cecilia , Lu Kristina T. , Lambris John D. , Zetoune Firas S. , Ward Peter A.
Published: April 2014

The complement-derived anaphylatoxin, C5a, is a potent phlogistic molecule that mediates its effects by binding to C5a receptor (C5aR; CD88). We now demonstrate specific binding of radiolabeled recombinant mouse C5a to mouse dermal microvascular endothelial cells (MDMEC) with a Kd50 of 3.6 nM and to ∼15,000–20,000 receptors/cell. Recombinant mC5a competed effectively with binding of [125I]rmC5a to MDMEC. Enhanced binding of C5a occurred, as well as increased mRNA for C5aR, after in vitro exposure of MDMEC to LPS, IFN-γ, or IL-6 in a time- and dose-dependent manner. By confocal microscopy, C5aR could be detected on surfaces of MDMEC using anti-C5aR Ab. In vitro expression of macrophage inflammatory protein-2 (MIP-2) and monocyte chemoattractant protein-1 (MCP-1) by MDMEC was also measured. Exposure of MDMEC to C5a or IL-6 did not result in changes in MIP-2 or MCP-1 production, but initial exposure of MDMEC to IL-6, followed by exposure to C5a, resulted in significantly enhanced production of MIP-2 and MCP-1 (but not TNF-α and MIP-1α). Although LPS or IFN-γ alone induced some release of MCP-1 and MIP-2, pre-exposure of these monolayers to LPS or IFN-γ, followed by addition of C5a, resulted in synergistic production of MIP-2 and MCP-1. Following i.v. infusion of LPS into mice, up-regulation of C5aR occurred in the capillary endothelium of mouse lung, as determined by immunostaining. These results support the hypothesis that C5aR expression on MDMEC and on the microvascular endothelium of lung can be up-regulated, suggesting that C5a in the co-presence of additional agonists may mediate pro-inflammatory effects of endothelial cells.
The T Cell Response to Art v 1, the Major Mugwort Pollen Allergen, Is Dominated by One Epitope
Authors: Jahn-Schmid Beatrice , Kelemen Peter , Himly Martin , Bohle Barbara , Fischer Gottfried , Ferreira Fatima , Ebner Christof
Published: April 2014

Mugwort (Artemisia vulgaris) pollen allergens represent the main cause of pollinosis in late summer in Europe. At least 95% of sera from mugwort pollen-allergic patients contain IgE against a highly glycosylated 24- to 28-kDa glycoprotein. Recently, this major allergen, termed Art v 1, was characterized, cloned in Escherichia coli, and produced in recombinant form. In the present study we characterized and compared the T cell responses to natural (nArt v 1) and recombinant Art v 1 (rArt v 1). In vitro T cell responses to nArt v 1 and rArt v 1 were studied in PBMC, T cell lines (TCL), and T cell clones (TCC) established from PBMC of mugwort-allergic patients. Stimulation of PBMC or allergen-specific TCL with either nArt v 1 or rArt v 1 resulted in comparable proliferative T cell responses. Eighty-five percent of the TCC reactive with rArt v 1 cross-reacted with the natural protein. The majority of the CD4+CD8−TCR αβ+ Art v 1-specific TCC, obtained from 10 different donors, belonged to the Th2 phenotype. Epitope mapping of TCL and TCC using overlapping peptides revealed a single immunodominant T cell epitope recognized by 81% of the patients. Inhibition experiments demonstrated that the presentation of this peptide is restricted by HLA-DR molecules. In conclusion, the T cell response to Art v 1 is characterized by one strong immunodominant epitope and evidently differs from the T cell responses to other common pollen allergens known to contain multiple T cell epitopes. Therefore, mugwort allergy may be an ideal candidate for a peptide-based immunotherapy approach.
Cutting Edge: Virus-Specific CD4+ Memory T Cells in Nonlymphoid Tissues Express a Highly Activated Phenotype
Authors: Cauley Linda S. , Cookenham Tres , Miller Timothy B. , Adams Pamela S. , Vignali Kate M. , Vignali Dario A. A. , Woodland David L.
Published: April 2014

Recent studies have shown that CD4+ memory T cells persist in nonlymphoid organs following infections. However, the development and phenotype of these peripheral memory cells are poorly defined. In this study, multimerized MHC-Ig fusion proteins, with a covalently attached peptide sequence from the Sendai virus hemagglutinin/neuraminidase gene, have been used to identify virus-specific CD4+ T cells during Sendai virus infection and the establishment of peripheral CD4+ memory populations in the lungs. We show declining frequencies of virus-specific CD4+ T cells in the lungs over the course of ∼3 mo after infection. Like peripheral CD8+ T cells, the CD4+ have an acutely activated phenotype, suggesting that a high level of differentiation is required to reach the airways and persist as memory cells. Differences in CD25 and CD11a expression indicate that the CD4+ cells from the lung airways and parenchyma are distinct memory populations.
Cutting Edge: A Novel Toll/IL-1 Receptor Domain-Containing Adapter That Preferentially Activates the IFN-β Promoter in the Toll-Like Receptor Signaling
Authors: Yamamoto Masahiro , Sato Shintaro , Mori Kiyotoshi , Hoshino Katsuaki , Takeuchi Osamu , Takeda Kiyoshi , Akira Shizuo
Published: April 2014

MyD88 is a Toll/IL-1 receptor (TIR) domain-containing adapter common to signaling pathways via Toll-like receptor (TLR) family. However, accumulating evidence demonstrates the existence of a MyD88-independent pathway, which may explain unique biological responses of individual TLRs, particularly TLR3 and TLR4. TIR domain-containing adapter protein (TIRAP)/MyD88 adapter-like, a second adapter harboring the TIR domain, is essential for MyD88-dependent TLR2 and TLR4 signaling pathways, but not for MyD88-independent pathways. Here, we identified a novel TIR domain-containing molecule, named TIR domain-containing adapter inducing IFN-β (TRIF). As is the case in MyD88 and TIRAP, overexpression of TRIF activated the NF-κB-dependent promoter. A dominant-negative form of TRIF inhibited TLR2-, TLR4-, and TLR7-dependent NF-κB activation. Furthermore, TRIF, but neither MyD88 nor TIRAP, activated the IFN-β promoter. Dominant-negative TRIF inhibited TLR3-dependent activation of both the NF-κB-dependent and IFN-β promoters. TRIF associated with TLR3 and IFN regulatory factor 3. These findings suggest that TRIF is involved in the TLR signaling, particularly in the MyD88-independent pathway.
Cutting Edge: Inflammatory Signals Drive Organ-Specific Autoimmunity to Normally Cross-Tolerizing Endogenous Antigen
Authors: Vezys Vaiva , Lefrançois Leo
Published: April 2014

Links have been observed between infections and the development of autoimmunity. Proposed explanations include activation of self-Ag-bearing APC. Using a model system in which transgenic OVA is expressed in enterocytes, we showed that CD8 T cell recognition of cross-presented Ag in gut-associated lymph nodes was tolerogenic. However, concomitant infection with vesicular stomatitis virus encoding OVA abrogated tolerance and induced disease. We now show that following transfer of naive OT-I T cells, the addition of wild-type vesicular stomatitis virus, oral cholera toxin, or CD40 triggering can induce intestinal disease in transgenic mice. Tissue damage accompanied dramatic increases in cytokine release by activated OT-I cells in the intestine. The data indicated that products of antigenically unrelated infections can combine with cross-presented self-Ags on APC to prime autoaggressiveness, independent of additional Ag release. These results help explain how diverse pathogens, lacking any homology to self-proteins, could be causative agents in induction of organ-specific autoimmunity.
Cutting Edge: A New Tool to Evaluate Human Pre-Erythrocytic Malaria Vaccines: Rodent Parasites Bearing a HybridPlasmodium falciparumCircumsporozoite Protein
Authors: Persson Cathrine , Oliveira Giane A. , Sultan Ali A. , Bhanot Purnima , Nussenzweig Victor , Nardin Elizabeth
Published: April 2014

Malaria vaccines containing the Plasmodium falciparum Circumsporozoite protein repeat domain are undergoing human trials. There is no simple method to evaluate the effect of vaccine-induced responses on P. falciparum sporozoite infectivity. Unlike the rodent malaria Plasmodium berghei, P. falciparum sporozoites do not infect common laboratory animals and only develop in vitro in human hepatocyte cultures. We generated a recombinant P. berghei parasite bearing P. falciparum Circumsporozoite protein repeats. These hybrid sporozoites are fully infective in vivo and in vitro. Monoclonal and polyclonal Abs to P. falciparum repeats neutralize hybrid parasite infectivity, and mice immunized with a P. falciparum vaccine are protected against challenge with hybrid sporozoites.
Macrophages and Dendritic Cells Use the Cytosolic Pathway to Rapidly Cross-Present Antigen from Live, Vaccinia-Infected Cells
Authors: Ramirez Maria Carmen , Sigal Luis J.
Published: April 2014

Professional APCs (pAPC) can process and present on their own MHC class I molecules Ags acquired from Ag donor cells (ADC). This phenomenon of cross-presentation is essential in the induction of CD8+ T cell responses to viruses that do not infect pAPC and possibly contributes to the induction of CD8+ responses to many other viruses. However, little is known about the mechanisms underlying this process. In this study, we show that dendritic cells and macrophages cross-present a model Ag supplied by vaccinia virus-infected ADC via the cytosolic route. Strikingly, we also found that cross-presentation of Ags provided by vaccinia-infected cells occurs within a couple of hours of pAPC/ADC interaction, that the duration of cross-presentation lasts for only 16 h, and that cross-presentation can occur at early times of infection when the ADC are still alive.
Phospholipase Cγ2 Is Essential for Specific Functions of FcεR and FcγR
Authors: Wen Renren , Jou Shiann-Tarng , Chen Yuhong , Hoffmeyer Angelica , Wang Demin
Published: April 2014

Phospholipase Cγ2 (PLCγ2) plays a critical role in the functions of the B cell receptor in B cells and of the FcRγ chain-containing collagen receptor in platelets. Here we report that PLCγ2 is also expressed in mast cells and monocytes/macrophages and is activated by cross-linking of FcεR and FcγR. Although PLCγ2-deficient mice have normal development and numbers of mast cells and monocytes/macrophages, we demonstrate that PLCγ2 is essential for specific functions of FcεR and FcγR. While PLCγ2-deficient mast cells have normal mitogen-activated protein kinase activation and cytokine production at mRNA levels, the mutant cells have impaired FcεR-mediated Ca2+ flux and inositol 1,4,5-trisphosphate production, degranulation, and cytokine secretion. As a physiological consequence of the effect of PLCγ2 deficiency, the mutant mice are resistant to IgE-mediated cutaneous inflammatory skin reaction. Macrophages from PLCγ2-deficient mice have no detectable FcγR-mediated Ca2+ flux; however, the mutant cells have normal FcγR-mediated phagocytosis. Moreover, PLCγ2 plays a nonredundant role in FcγR-mediated inflammatory skin reaction.
Regulation of N-Formyl Peptide-Mediated Degranulation by Receptor Phosphorylation
Authors: Vines Charlotte M. , Xue Mei , Maestas Diane C. , Cimino Daniel F. , Prossnitz Eric R.
Published: April 2014

One of the major functions of the N-formyl peptide receptor (FPR) is to mediate leukocyte degranulation. Phosphorylation of the C-terminal domain of the FPR is required for receptor internalization and desensitization. Although arrestins mediate phosphorylation-dependent desensitization, internalization, and initiation of novel signaling cascades for a number of G protein-coupled receptors, their roles in FPR regulation and signaling remain unclear. CXCR1-mediated degranulation of RBL-2H3 cells is promoted by arrestin binding. To determine whether receptor phosphorylation or arrestin binding is required to promote FPR-mediated degranulation, we used RBL-2H3 cells stably transfected with either the wild-type FPR or a mutant form, ΔST, which is incapable of undergoing ligand-stimulated phosphorylation. We observed that stimulation of wild-type FPR resulted in very low levels of degranulation compared with that mediated by cross-linking of the FcεRI receptor. Stimulation of the ΔST mutant, however, resulted in levels of degranulation comparable to those of the FcεRI receptor, demonstrating that neither receptor phosphorylation nor arrestin binding was necessary to initiate FPR-mediated degranulation. Degranulation initiated by the ΔST mutant was proportional to the level of active cell surface receptor, suggesting that either receptor internalization or desensitization may be responsible for terminating degranulation of the wild-type FPR. To distinguish between these possibilities, we used a partially phosphorylation-deficient mutant of the FPR that can undergo internalization, but not desensitization. Degranulation by this mutant FPR was indistinguishable from that of the ΔST mutant, indicating that FPR phosphorylation or binding of arrestin but not internalization terminates the degranulation response.
Lyn and Syk Kinases Are Sequentially Engaged in Phagocytosis Mediated by FcγR
Authors: Strzelecka-Kiliszek Agnieszka , Kwiatkowska Katarzyna , Sobota Andrzej
Published: April 2014

Recent data indicate that phagocytosis mediated by FcγRs is controlled by the Src and Syk families of protein tyrosine kinases. In this study, we demonstrate a sequential involvement of Lyn and Syk in the phagocytosis of IgG-coated particles. The particles isolated at the stage of their binding to FcγRs (4°C) were accompanied by high amounts of Lyn, in addition to the signaling γ-chain of FcγRs. Simultaneously, the particle binding induced rapid tyrosine phosphorylation of numerous proteins. During synchronized internalization of the particles induced by shifting the cell to 37°C, Syk kinase and Src homology 2-containing tyrosine phosphatase-1 (SHP-1) were associated with the formed phagosomes. At this step, most of the proteins were dephosphorylated, although some underwent further tyrosine phosphorylation. Quantitative immunoelectron microscopy studies confirmed that Lyn accumulated under the plasma membrane beneath the bound particles. High amounts of the γ-chain and tyrosine-phosphorylated proteins were also observed under the bound particles. When the particles were internalized, the γ-chain was still detected in the region of the phagosomes, while amounts of Lyn were markedly reduced. In contrast, the vicinity of the phagosomes was heavily decorated with anti-Syk and anti-SHP-1 Abs. The local level of protein tyrosine phosphorylation was reduced. The data indicate that the accumulation of Lyn during the binding of IgG-coated particles to FcγRs correlated with strong tyrosine phosphorylation of numerous proteins, suggesting an initiating role for Lyn in protein phosphorylation at the onset of the phagocytosis. Syk kinase and SHP-1 phosphatase are mainly engaged at the stage of particle internalization.
WY14,643, a PPARα Ligand, Has Profound Effects on Immune Responses In Vivo
Authors: Cunard Robyn , DiCampli Dennis , Archer D. Clay , Stevenson Jennifer L. , Ricote Mercedes , Glass Christopher K. , Kelly Carolyn J.
Published: April 2014

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors with diverse actions. PPARα and PPARγ are expressed in different lymphocyte subpopulations. Recently, we have observed that PPARα ligands elicit augmented IL-4 expression in cultures of mitogen-activated splenocytes. The following studies were undertaken to characterize the in vivo effects of WY14,643, a PPARα ligand. Our studies demonstrate that oral administration of WY14,643 markedly reduces splenocyte number in immunized and nonimmunized C57BL/6 mice. Mice fed WY14,643 display impaired IgG responses to myelin oligodendrocyte glycoprotein peptide 35–55 (pMOG35–55), following immunization with pMOG35–55/CFA. Following in vitro restimulation with pMOG35–55, splenocytes harvested from WY14,643-fed mice demonstrate impaired production of IFN-γ, IL-6, and TNF-α despite similar proliferative responses. We also demonstrate higher expression of PPARα in B than T cells. Finally, to obtain an understanding of the cause of splenocyte depletion with fibrate therapy, we studied the effect of WY14,643 on apoptosis of activated splenocytes. WY14,643 in vitro induces apoptosis in lymphocytes and this effect appears to occur in a PPARα-independent manner. Thus WY14,643, a fibrate, is a profound immunosuppressive agent.
Peroxisome Proliferator-Activated Receptor γ-Mediated NF-κB Activation and Apoptosis in Pre-B Cells
Authors: Schlezinger Jennifer J. , Jensen Brenda A. , Mann Koren K. , Ryu Heui-Young , Sherr David H.
Published: April 2014

The role of peroxisome proliferator-activated receptor γ (PPARγ) in adipocyte physiology has been exploited for the treatment of diabetes. The expression of PPARγ in lymphoid organs and its modulation of macrophage inflammatory responses, T cell proliferation and cytokine production, and B cell proliferation also implicate it in immune regulation. Despite significant human exposure to PPARγ agonists, little is known about the consequences of PPARγ activation in the developing immune system. Here, well-characterized models of B lymphopoiesis were used to investigate the effects of PPARγ ligands on nontransformed pro/pre-B (BU-11) and transformed immature B (WEHI-231) cell development. Treatment of BU-11, WEHI-231, or primary bone marrow B cells with PPARγ agonists (ciglitazone and GW347845X) resulted in rapid apoptosis. A role for PPARγ and its dimerization partner, retinoid X receptor (RXR)α, in death signaling was supported by 1) the expression of RXRα mRNA and cytosolic PPARγ protein, 2) agonist-induced binding of PPARγ to a PPRE, and 3) synergistic increases in apoptosis following cotreatment with PPARγ agonists and 9-cis-retinoic acid, an RXRα agonist. PPARγ agonists activated NF-κB (p50, Rel A, c-Rel) binding to the upstream κB regulatory element site of c-myc. Only doses of agonists that induced apoptosis stimulated NF-κB-DNA binding. Cotreatment with 9-cis-retinoic acid and PPARγ agonists decreased the dose required to activate NF-κB. These data suggest that activation of PPARγ-RXR initiates a potent apoptotic signaling cascade in B cells, potentially through NF-κB activation. These results have implications for the nominal role of the PPARγ in B cell development and for the use of PPARγ agonists as immunomodulatory therapeutics.
The Roles of IL-12 in Providing a Third Signal for Clonal Expansion of Naive CD8 T Cells
Authors: Valenzuela Javier , Schmidt Clint , Mescher Matthew
Published: April 2014

Stimulation of an effective in vitro or in vivo response by naive CD8 T cells requires three signals: TCR engagement, costimulation/IL-2, and a third signal that can be provided by IL-12. In addition to being required for acquisition of cytolytic function, IL-12 is required for optimal IL-2-dependent proliferation and clonal expansion. In experiments examining in vitro stimulation of naive CD8 T cells, IL-12 is shown to stimulate expression of the IL-2R α-chain (CD25) to much higher levels than are reached in response to just TCR and costimulation and/or IL-2. In addition, high CD25 expression is substantially prolonged in the presence of IL-12. As a consequence, the cells proliferate more effectively in response to low levels of IL-2. Examination of adoptively transferred TCR transgenic CD8 T cells responding to peptide Ag confirmed that IL-12 up-regulates CD25 in vivo, even when B7-mediated costimulation is largely blocked. TCR- and IL-2-dependent proliferation of CD8 T cells from mice deficient in CD25 was also found to increase in the presence of IL-12, indicating that CD25 up-regulation is not the only mechanism by which IL-12 increases clonal expansion of the cells. IL-2 and IL-12 both act to increase expression of both CD25 and the IL-12R, thus providing positive cross-regulation of receptor expression. These results suggest that when cross-priming dendritic cells present class I/Ag and costimulatory ligands, and produce IL-12, naive CD8 T cells will begin to produce IL-2 and both receptors will be optimally up-regulated to insure that an effective response is generated.
B Cell Selection and Affinity Maturation During an Antibody Response in the Mouse with Limited B Cell Diversity
Authors: Kanayama Naoki , Kimoto Takafumi , Todo Kagefumi , Nishikawa Yumiko , Hikida Masaki , Magari Masaki , Cascalho Marilia , Ohmori Hitoshi
Published: April 2014

The quasi-monoclonal mouse has limited B cell diversity, whose major (∼80%) B cell Ag receptors are comprised of the knockin VH 17.2.25 (VHT)-encoded H chain and the λ1 or λ2 L chain, thereby being specific for 4-hydroxy-3-nitrophenylacetyl. The p-nitrophenylacetyl (pNP) was found to be a low affinity analog of nitrophenylacetyl. We examined affinity maturation of anti-pNP IgG by analyzing mAbs obtained from quasi-monoclonal mice that were immunized with this low affinity Ag. The results are: 1) Although VHT/λ1 and VHT/λ2 IgM were equally produced, VHT/λ2 IgG almost exclusively underwent affinity maturation toward pNP. 2) A common mutation in complementarity-determining region 3 of VHT (T313A) mainly contributed to generating the specificity for pNP. 3) Because mutated VHT-encoded γ-chains could form λ1-bearing IgG in Chinese hamster ovary cells, apparent absence of VHT/λ1 anti-pNP IgG may not be due to the incompatibility between the γ-chains and the λ1-chain, but may be explained by the fact that VHT/λ1 B cells showed 50- to 100-fold lower affinity for pNP than VHT/λ2 B cells. 4) Interestingly, a pNP-specific IgM mAb that shared common mutations including T313A with high affinity anti-pNP IgG was isolated, suggesting that a part of hypermutation coupled with positive selection can occur before isotype switching. Thus, even weak B cell receptor engagement can elicit an IgM response, whereas only B cells that received signals stronger than a threshold may be committed to an affinity maturation process.
Hyperconservation of the Putative Antigen Recognition Site of the MHC Class I-b Molecule TL in the Subfamily Murinae: Evidence That Thymus Leukemia Antigen Is an Ancient Mammalian Gene
Authors: Davis Beckley K. , Cook Richard G. , Rich Robert R. , Rodgers John R.
Published: April 2014

“Classical” MHC class I (I-a) genes are extraordinarily polymorphic, but “nonclassical” MHC class I (I-b) genes are monomorphic or oligomorphic. Although diversifying (positive) Darwinian selection is thought to explain the origin and maintenance of MHC class I-a polymorphisms, genetic mechanisms underlying MHC class I-b evolution are uncertain. In one extreme model, MHC class I-b loci are derived by gene duplication from MHC class I-a alleles but rapidly drift into functional obsolescence and are eventually deleted. In this model, extant MHC class I-b genes are relatively young, tend to be dysfunctional or pseudogenic, and orthologies are restricted to close taxa. An alternative model proposed that the mouse MHC class I-b gene thymus leukemia Ag (TL) arose ∼100 million years ago, near the time of the mammalian radiation. To determine the mode of evolution of TL, we cloned TL from genomic DNA of 11 species of subfamily Murinae. Every sample we tested contained TL, suggesting this molecule has been maintained throughout murine evolution. The sequence similarity of TL orthologs ranged from 85–99% and was inversely proportional to taxonomic distance. The sequences showed high conservation throughout the entire extracellular domains with exceptional conservation in the putative Ag recognition site. Our results strengthen the hypotheses that TL has evolved a specialized function and represents an ancient MHC class I-b gene.
HLA-DP4, the Most Frequent HLA II Molecule, Defines a New Supertype of Peptide-Binding Specificity
Authors: Castelli Florence A. , Buhot Cécile , Sanson Alain , Zarour Hassane , Pouvelle-Moratille Sandra , Nonn Céline , Gahery-Ségard Hanne , Guillet Jean-Gérard , Ménez André , Georges Bertrand , Maillère Bernard
Published: April 2014

Among HLA-DP specificities, HLA-DP4 specificity involves at least two molecules, HLA-DPA1*0103/DPB1*0401 (DP401) and HLA-DPA1*0103/DPB1*0402 (DP402), which differ from each other by only three residues. Together, they are present worldwide at an allelic frequency of 20–60% and are the most abundant human HLA II alleles. Strikingly, the peptide-binding specificities of these molecules have never been investigated. Hence, in this study, we report the peptide-binding motifs of both molecules. We first set up a binding assay specific for the immunopurified HLA-DP4 molecules. Using multiple sets of synthetic peptides, we successfully defined the amino acid preferences of the anchor residues. With these assays, we were also able to identify new peptide ligands from allergens and viral and tumor Ags. DP401 and DP402 exhibit very similar patterns of recognition in agreement with molecular modeling of the complexes. Pockets P1 and P6 accommodate the main anchor residues and interestingly contain only two polymorphic residues, β86 and β11, respectively. Both positions are almost dimorphic and thus produce a limited number of pocket combinations. Taken together, our results support the existence of three main binding supertypes among HLA-DP molecules and should significantly contribute to the identification of universal epitopes to be used in peptide-based vaccines for cancer, as well as for allergic or infectious diseases.
L-MBP Is Expressed in Epithelial Cells of Mouse Small Intestine
Authors: Uemura Kazuhide , Saka Masako , Nakagawa Tomoaki , Kawasaki Nobuko , Thiel Steffen , Jensenius Jens Christian , Kawasaki Toshisuke
Published: April 2014

The mannan-binding proteins (L-MBP and S-MBP, also denoted MBL-C and MBL-A), mainly produced in liver and existing in liver and serum, play important roles in the innate immunity against a variety of pathogens. Total RNA from mouse tissues were screened for MBP mRNA by RT-PCR. In addition to liver, S-MBP mRNA was detected in lung, kidney, and testis, and L-MBP mRNA was detected in kidney, thymus, and small intestine. Quantitative RT-PCR revealed that the small intestine is a predominant site of extrahepatic expression of L-MBP. Western blotting with polyclonal Abs against rat L-MBP demonstrated this protein in Triton X-100 extracts of the small intestine obtained from mice that had undergone systemic perfusion. Immunohistochemical staining with an mAb against mouse L-MBP and in situ hybridization revealed that L-MBP is selectively expressed in some villous epithelial cells of the small intestine. These findings suggest that L-MBP plays a role in mucosal innate immunity.