bluuebunny/crossref_metadata_2025 huggingface dataset:
IFN-β Differentially Regulates CD40-Induced Cytokine Secretion by Human Dendritic Cells
Authors: McRae Bradford L. , Beilfuss Beth A. , Seventer Gijs A. van
Published: April 2014

We have previously shown that IFN-β, a key cytokine associated with the early phase of the innate host defense, can prevent the generation of human Th1 cells. Specifically, we demonstrated that IFN-β prevents the in vitro monocyte-derived mature dendritic cell (DC)-dependent differentiation of naive Th cells into IFN-γ-secreting Th cells, as a result of its ability to inhibit DC IL-12 secretion. The goal of the present study was to identify how IFN-β negatively regulates IL-12 secretion by DC. We report that in our Th cell differentiation model, DC IL-12 secretion is dependent on the CD40L/CD40 accessory pathway, and, utilizing a Th cell-free system, we find that IFN-β inhibits anti-CD40 mAb-induced DC secretion of the p40 chain of the IL-12 heterodimer. In addition, we show that IFN-β-mediated inhibition of CD40 signaling does not interfere with all signaling pathways emanating from CD40, since anti-CD40 mAb-induced DC IL-6 secretion is augmented by IFN-β. Thus, our results demonstrate that signaling from CD40 is differentially regulated by IFN-β. A second critical element of innate immunity involves the response against components of bacterial membranes such as LPS. DC respond to LPS by secreting IL-6 and IL-12. In contrast to CD40-dependent IL-6 and IL-12 secretion, we find that LPS-induced DC secretion of p40 IL-12 and IL-6 is not affected by IFN-β. Our findings show that IFN-β influences the generation of acquired immune responses through its regulation of CD40-dependent DC functions.
A Role for Pref-1 and HES-1 in Thymocyte Development
Authors: Kaneta Midori , Osawa Masatake , Osawa Mitsujiro , Sudo Kazuhiro , Nakauchi Hiromitsu , Farr Andrew G. , Takahama Yousuke
Published: April 2014

T lymphocyte development requires a series of interactions between developing thymocytes and thymic epithelial (TE) cells. In this paper we show that TE cells in the developing thymus express Pref-1, a Delta-like cell-surface molecule. In fetal thymus organ cultures (FTOC), thymocyte cellularity was increased by the exogenous dimeric Pref-1 fusion protein, but was reduced by the soluble Pref-1 monomer or anti-Pref-1 Ab. Dimeric Pref-1 in FTOC also increased thymocyte expression of the HES-1 transcription factor. Thymocyte cellularity was increased in FTOC repopulated with immature thymocytes overexpressing HES-1, whereas FTOC from HES-1-deficient mice were hypocellular and unresponsive to the Pref-1 dimer. We detected no effects of either Pref-1 or HES-1 on developmental choice among thymocyte lineages. These results indicate that Pref-1 expressed by TE cells and HES-1 expressed by thymocytes are critically involved in supporting thymocyte cellularity.
Importance of the Membrane-Proximal Extracellular Domains for Activation of the Signal Transducer Glycoprotein 130
Authors: Kurth Ingo , Horsten Ursula , Pflanz Stefan , Timmermann Andreas , Küster Andrea , Dahmen Heike , Tacken Ingrid , Heinrich Peter C. , Müller-Newen Gerhard
Published: April 2014

The transmembrane glycoprotein gp130 is the common signal transducing receptor subunit of the IL-6-type cytokines. The gp130 extracellular part is predicted to consist of six individual domains. Whereas the role of the three membrane-distal domains (D1–D3) in binding of IL-6 and IL-11 is well established, the function of the membrane-proximal domains (D4–D6) is unclear. Mapping of a neutralizing mAb to the membrane-proximal part of gp130 suggests a functional role of D4–D6 in receptor activation. Individual deletion of these three domains differentially interferes with ligand binding of the soluble and membrane-bound receptors. All deletion mutants do not signal in response to IL-6 and IL-11. The deletion mutants Δ4 and, to a lesser extent, Δ6 are still activated by agonistic monoclonal gp130 Abs, whereas the deletion mutant Δ5 does not respond. Because membrane-bound Δ5 binds IL-6/soluble IL-6R as does wild-type gp130, but does not transduce a signal in response to various stimuli, this domain plays a prominent role in coupling of ligand binding and signal transduction. Replacement of the fifth domain of gp130 by the corresponding domain of the homologous G-CSF receptor leads to constitutive activation of the chimera upon overexpression in COS-7 cells. In HepG2 cells this mutant responds to IL-6 comparable to wild-type gp130. Our findings suggest a functional role of the membrane-proximal domains of gp130 in receptor activation. Thus, within the hematopoietic receptor family the mechanism of receptor activation critically depends on the architecture of the receptor ectodomain.
Limited Diversity of Peptides Related to an Alloreactive T Cell Epitope in the HLA-B27-Bound Peptide Repertoire Results from Restrictions at Multiple Steps Along the Processing-Loading Pathway
Authors: Paradela Alberto , Alvarez Iñaki , García-Peydró Marina , Sesma Laura , Ramos Manuel , Vázquez Jesús , López de Castro José A.
Published: April 2014

The influence of various factors along the processing-loading pathway in limiting the diversity of HLA-B27-bound peptides around a core protein sequence was analyzed. The C5 proteasome subunit-derived RRFFPYYV and RRFFPYYVY peptides are natural B*2705 ligands. The octamer is an allospecific CTL epitope. Digestion of a 27-mer fragment of C5 revealed that both ligands are generated from this precursor substrate with the 20S proteasome in vitro in a ratio comparable to that in the B*2705-bound peptide pool. The C5 sequence allowed to derive a nested set of six additional peptides with 8–11 residues containing the core octamer sequence and the Arg2 motif of HLA-B27, none of which was found in the B27-bound pool. Together, low proteasomal yield, disfavored TAP-binding motifs, and low affinity for B*2705 accounted for the absence of four of the six peptides. The two remaining differed from the natural octamer or nonamer ligands only by an additional N-terminal Ser residue. Their stability in complex with B*2705 was lower than the respective natural ligands, raising the possibility that N-terminal trimming might have favored a shift toward the more stable peptides. The results suggest that the B*2705-bound peptide repertoire has a highly restricted diversity around a core alloantigenic sequence. This is not explained by a single bottleneck feature, but by multiple factors, including proteasomal generation, TAP-binding motifs, MHC-binding efficiency, and perhaps optimized stability through N-terminal trimming. Tapasin-dependent restrictions, although not excluded, were not required to explain the absence in vivo of the particular peptide set in this study.
Point Mutation of a Tyrosine in the Linker Region of Syk Results in a Gain of Function
Authors: Sada Kiyonao , Zhang Juan , Siraganian Reuben P.
Published: April 2014

The protein tyrosine kinase Syk plays an essential role in FcεRI-mediated histamine release in mast cells by regulating the phosphorylation of other proteins. We investigated the functional role of a putative Syk phosphorylation site, Tyr317. This tyrosine in the linker region of Syk is a possible site for binding by the negative regulator Cbl. Syk with Tyr317 mutated to Phe (Y317F) was expressed in a Syk-negative variant of the RBL-2H3 mast cells. Compared with cells expressing wild-type Syk, expression of the Y317F mutant resulted in an increase in the FcεRI-mediated tyrosine phosphorylation of phospholipase C-γ and a dramatic enhancement of histamine release. The in vivo FcεRI-induced tyrosine phosphorylation of wild-type Syk and that of the Y317F mutant were similar. Although the FcεRI-induced tyrosine phosphorylation of total cellular proteins was enhanced in the cells expressing the Y317F Syk, the phosphorylation of some other molecules, including the receptor subunits, Vav and mitogen-activated protein kinase, was not increased. The FcεRI-induced phosphorylation of Cbl was downstream of Syk kinase activity and was unchanged by expression of the Y317F mutation. These data indicate that Tyr317 in the linker region of Syk functions to negatively regulate the signals leading to degranulation.
Specific Inhibition of Cyclooxygenase 2 Restores Antitumor Reactivity by Altering the Balance of IL-10 and IL-12 Synthesis
Authors: Stolina Marina , Sharma Sherven , Lin Ying , Dohadwala Mariam , Gardner Brian , Luo Jie , Zhu Li , Kronenberg Mitchell , Miller Patrice W. , Portanova Joseph , Lee Jason C. , Dubinett Steven M.
Published: April 2014

Cyclooxygenase-2 (COX-2), the enzyme at the rate-limiting step of prostanoid production, has been found to be overexpressed in human lung cancer. To evaluate lung tumor COX-2 modulation of antitumor immunity, we studied the antitumor effect of specific genetic or pharmacological inhibition of COX-2 in a murine Lewis lung carcinoma (3LL) model. Inhibition of COX-2 led to marked lymphocytic infiltration of the tumor and reduced tumor growth. Treatment of mice with anti-PGE2 mAb replicated the growth reduction seen in tumor-bearing mice treated with COX-2 inhibitors. COX-2 inhibition was accompanied by a significant decrement in IL-10 and a concomitant restoration of IL-12 production by APCs. Because the COX-2 metabolite PGE2 is a potent inducer of IL-10, it was hypothesized that COX-2 inhibition led to antitumor responses by down-regulating production of this potent immunosuppressive cytokine. In support of this concept, transfer of IL-10 transgenic T lymphocytes that overexpress IL-10 under control of the IL-2 promoter reversed the COX-2 inhibitor-induced antitumor response. We conclude that abrogation of COX-2 expression promotes antitumor reactivity by restoring the balance of IL-10 and IL-12 in vivo.
Development of Lupus in BXSB Mice Is Independent of IL-4
Authors: Kono Dwight H. , Balomenos Dimitrios , Park Miyo S. , Theofilopoulos Argyrios N.
Published: April 2014

Although systemic lupus erythematosus appears to be a humorally mediated disease, both Th1 and Th2 type responses have been implicated in its pathogenesis. The Th1 response, as exemplified by IFN-γ production, has been uniformly shown in mouse lupus models to be critical for disease induction. The role of Th2 type responses, however, is more complicated, with some studies showing detrimental and others beneficial effects of IL-4 in these models. To further address this issue, we generated and analyzed IL-4 gene-deficient BXSB mice. Mice homozygous for this deletion had significantly lower serum levels of total IgG1 compared with wild-type BXSB, consistent with the lack of IL-4. However, no significant differences were observed in mortality, spleen weight, severity of glomerulonephritis, levels of anti-chromatin and anti-ssDNA Abs, or frequency of activated (CD44high) CD4+ T cells. The anti-chromatin Ab isotype response was virtually all Th1 type in both the knockout and wild-type BXSB. These findings directly demonstrate that IL-4 and, by inference, Th2 cells are not obligatory participants in the induction and maintenance of lupus in this strain.
Differential Contribution of Fas- and Perforin-Mediated Mechanisms to the Cell-Mediated Cytotoxic Activity of Naive and In Vivo-Primed Intestinal Intraepithelial Lymphocytes
Authors: Corazza Nadia , Müller Stefan , Brunner Thomas , Kägi David , Mueller Christoph
Published: April 2014

Intestinal intraepithelial lymphocytes (IELs) are known to exert strong constitutive cytotoxic activity. In the present study we compared the Ag-specific cytotoxic activity and the effector mechanisms involved in non-Ag-primed, naive and in in vivo-primed IELs and splenic CD8 T cells. Ex vivo isolated naive CD8αα TCRαβ IELs, CD8αβ IELs, and splenocytes from lymphocytic choriomeningitis virus (LCMV)-specific TCR transgenic mice exert Ag-specific cytotoxic activity in a long-term, but not in a short-term, cytotoxicity assay. This cytotoxic activity is mainly Fas-Fas ligand mediated and is significantly reduced in the presence of 20 μg/ml Fas-Fcγ1 fusion protein. Both CD8αβ IELs and CD8αβ splenocytes isolated from LCMV-infected C57BL/6 mice exert potent perforin-dependent cell-mediated cytotoxicity. CD8αα TCRαβ IELs from LCMV-infected animals, however, show only minimal Ag-specific cytotoxicity. The potent cytotoxic activity of in vivo activated CD8αβ IELs is not affected by the addition of Fas-Fcγ1. Nevertheless CD8αβ IELs from LCMV-infected perforin-deficient mice exert Ag-specific cytotoxicity in a short-term cytotoxicity assay, and this cytotoxicity is almost completely blocked by the addition of Fas-Fcγ1. These results demonstrate that naive CD8αβ IELs exert Ag-specific, Fas-Fas ligand-mediated, constitutive cytotoxic activity in a long-term cytotoxicity assay, whereas primed CD8αβ IELs primarily use the perforin-dependent exocytosis pathway to exert their potent cytotoxic activity. Furthermore, these results clearly illustrate the requirement for Ag-specific determination of IEL-mediated cytotoxicity, because the elevated, but variable, frequencies of memory-type T cells in this compartment may lead to ambiguous results when polyclonal activation or redirected assays are used.
fMet-Leu-Phe Stimulates Proinflammatory Cytokine Gene Expression in Human Peripheral Blood Monocytes: The Role of Phosphatidylinositol 3-Kinase
Authors: Pan Zhixing K. , Chen Ling-Yu , Cochrane Charles G. , Zuraw Bruce L.
Published: April 2014

The fMLP-stimulated release of proinflammatory cytokines such as IL-1 by human peripheral blood monocytes is an important component of the inflammatory process. The signaling mechanisms used by fMLP to stimulate the release of cytokines are still incompletely understood. We previously demonstrated that fMLP-stimulated NF-κB activation in PBMC and now we present evidence that the lipid products of phosphatidylinositol 3-kinase (PI 3-kinase) are required for fMLP-stimulated activation of NF-κB. Pretreatment with the PI 3-kinase inhibitors, wortmannin and LY294002, effectively blocked fMLP-induced IL-1β gene expression as well as NF-κB activation. Transient transfection of THP1 cells with a dominant-negative mutant of the PI 3-kinase p85 subunit also abrogated fMLP-induced κB activity. These results suggest a potential role of fMLP in the transcription of proinflammatory cytokines and provide the first evidence that such regulation may occur through PI 3-kinase activity.
Role of B7-CD28/CTLA-4 Costimulation and NF-κB in Allergen-Induced T Cell Chemotaxis by IL-16 and RANTES
Authors: Hidi Rabia , Riches Vanessa , Al-Ali Musa , Cruikshank William W. , Center David M. , Holgate Stephen T. , Djukanović Ratko
Published: April 2014

The mechanisms that cause T cell recruitment into inflamed airways of asthmatic individuals are poorly understood. It has been shown previously that both natural exposure to allergen and challenge in the laboratory induce T cell accumulation in the bronchial mucosa of sensitized asthmatics. To study the mechanisms involved in this process, we have used an explant model in which bronchial biopsies taken from mild atopic asthmatic volunteers during fiberoptic bronchoscopy were stimulated in culture for 24 h by the common aeroallergen house dust mite (Dermatophagoides pteronyssinus (Der p)). Analysis of culture supernatants showed that stimulation with Der p significantly enhanced both the generation of T cell chemotactic activity by the mucosal tissue, as assayed in microchemotaxis chambers, and the production of IL-16 and RANTES. Neutralization experiments showed that IL-16 contributed more to the chemotactic activity than RANTES. The fusion protein CTLA-4-Ig, blocking B7:CD28 costimulation, and dexamethasone both significantly reduced the ex vivo production of chemotactic activity and release of IL-16 and RANTES. The proteasome inhibitor Cbz-Ile-Glu(OtBu)-Ala-leucinal also had a significant inhibitory effect on T cell chemotactic activity and IL-16 but not RANTES generation, indicating a role for nuclear factor NFκB activation. These results indicate that allergen stimulates cells within the bronchial mucosa to increase IL-16 and RANTES release, both of which contribute to T cell accumulation in asthmatic airways. The allergen-induced chemotactic activity is dependent on cell activation via CD28 and involves, at least partly, NF-κB.
Monocyte Migration Through the Alveolar Epithelial Barrier: Adhesion Molecule Mechanisms and Impact of Chemokines
Authors: Rosseau Simone , Selhorst Jochen , Wiechmann Kristina , Leissner Katja , Maus Ulrich , Mayer Konstantin , Grimminger Friedrich , Seeger Werner , Lohmeyer Jürgen
Published: April 2014

Alveolar monocyte influx requires adherence and transmigration through the vascular endothelium, extracellular matrix, and alveolar epithelium. For investigating the monocyte migratory process across the epithelial barrier, we employed both the A549 cell line and isolated human alveolar epithelial cells. Under baseline conditions, spontaneous bidirectional transepithelial monocyte migration was noted, which was dose-dependently increased in the presence of the monocyte chemoattractant protein-1. TNF-α stimulation of the alveolar epithelium provoked the polarized apical secretion of monocyte chemoattractant protein-1 and RANTES and up-regulation of ICAM-1 and VCAM-1 expression, accompanied by markedly enhanced transepithelial monocyte traffic in the basal-to-apical direction. Multiple adhesive interactions were noted to contribute to the enhanced monocyte traffic across the TNF-α-stimulated alveolar epithelium: these included the β2 integrins CD11a, CD11b, CD11c/CD18, the β1 integrins very late Ag (VLA)-4, -5, and -6, and the integrin-associated protein CD47 on monocytes, as well as ICAM-1, VCAM-1, CD47, and matrix components on the epithelial side. In contrast, spontaneous monocyte migration through unstimulated epithelium depended predominantly on CD11b/CD18 and CD47, with some additional contribution of VLA-4, -5, and -6. In summary, unlike transendothelial monocyte traffic, for which β1 and β2 integrins are alternative mechanisms, monocyte migration across the alveolar epithelium largely depends on CD11b/CD18 and CD47 but required the additional engagement of the β1 integrins for optimal migration. In response to inflammatory challenge, the alveolar epithelium orchestrates enhanced monocyte traffic to the apical side by polarized chemokine secretion and up-regulation of ICAM-1 and VCAM-1.
The Arthus Reaction in Rodents: Species-Specific Requirement of Complement
Authors: Szalai Alexander J. , Digerness Stanley B. , Agrawal Alok , Kearney John F. , Bucy R. Pat , Niwas Shri , Kilpatrick John M. , Babu Y. Sudhakara , Volanakis John E.
Published: April 2014

We induced reverse passive Arthus (RPA) reactions in the skin of rodents and found that the contribution of complement to immune complex-mediated inflammation is species specific. Complement was found to be necessary in rats and guinea pigs but not in C57BL/6J mice. In rats, within 4 h after initiation of an RPA reaction, serum alternative pathway hemolytic titers decreased significantly below basal levels, whereas classical pathway titers were unchanged. Thus the dermal reaction proceeds coincident with systemic activation of complement. The serine protease inhibitor BCX 1470, which blocks the esterolytic and hemolytic activities of the complement enzymes Cls and factor D in vitro, also blocked development of RPA-induced edema in the rat. These data support the proposal that complement-mediated processes are of major importance in the Arthus reaction in rats and guinea pigs, and suggest that BCX 1470 will be useful as an anti-inflammatory agent in diseases where complement activation is known to be detrimental.
Cutting Edge: Tyrosine-Independent Transmission of Inhibitory Signals by CTLA-4
Authors: Cinek Tomas , Sadra Ali , Imboden John B.
Published: April 2014

CTLA-4 is an important inhibitor of T cell activation. We used Jurkat cells expressing mutants of murine CTLA-4 to study the structural requirements for inhibitory signaling. We find that signals for the inhibition of IL-2 secretion are delivered efficiently by a CTLA-4 mutant in which both cytoplasmic tyrosines have been replaced by phenylalanines. A CTLA-4 mutant that lacks the carboxyl-terminal half of the intracellular domain also retains the ability to inhibit, but deletion of an additional 11 aa completely abrogates that capability. We conclude that delivery of an inhibitory signal requires the membrane-proximal region of the CTLA-4 cytoplasmic domain and does not depend upon the tyrosine phosphorylation of CTLA-4.
Synergistic Effects of IL-4 and IL-18 on IL-12-Dependent IFN-γ Production by Dendritic Cells
Authors: Fukao Taro , Matsuda Satoshi , Koyasu Shigeo
Published: April 2014

Mouse splenic dendritic cells (DCs) produce IFN-γ in response to IL-12. In the present study, we analyzed effects of Th1 and Th2 cytokines on IFN-γ production by DCs. IL-18 produced by DCs and macrophages acts in an autocrine manner and augments IL-12-induced IFN-γ production by DCs as also observed in T and NK cells. Surprisingly, IL-4, a Th2 cytokine, also acts synergistically with IL-12 on IFN-γ production by DCs. In addition, IL-4 markedly enhances IFN-γ production when DCs are stimulated through CD40 or MHC class II. These results indicate that both Th1 and Th2 cytokines act on DCs during T cell-DC interaction upon Ag presentation. p38 mitogen-activated protein kinase is constitutively activated in mature DCs and is required for IFN-γ production by DCs. IL-18 but not IL-4 or IL-12 further activates the p38 mitogen-activated protein kinase activity, suggesting that IL-4 and IL-18 enhance IFN-γ production through distinct intracellular signal transduction pathways in DCs.
Differential Requirement for CD80 and CD80/CD86-Dependent Costimulation in the Lung Immune Response to an Influenza Virus Infection
Authors: Lumsden Joanne M. , Roberts Joanna M. , Harris Nicola L. , Peach Robert J. , Ronchese Franca
Published: April 2014

The CD28 costimulatory pathway is critical to T cell activation. Blockade of the interaction of CD28 with its ligands CD80 and CD86 using CTLA4-Ig has been proposed as a therapy for a number of immune-based disorders. We have used a murine model of influenza virus infection to study the role of CD28-dependent costimulation in the development of antiviral immune responses. In vivo treatment with CTLA4-Ig to block the interaction of CD28 with CD80 and CD86 reduced virus-specific cytotoxicity and IFN-γ production by bronchoalveolar lavage fluid CD8+ T lymphocytes in vitro. It also resulted in decreased numbers of virus-specific CD8+ T lymphocytes in the bronchoalveolar lavage fluid, lung, and spleen and lowered virus-specific Ab titers. Mice treated with CTLA4-Ig were able to control and clear the virus infection, but this was delayed compared with controls. Treatment with Y100F-Ig, a mutant form of CTLA4-Ig which selectively binds to CD80 and blocks the CD28-CD80 interaction leaving CD28-CD86 binding intact, did not affect Ab production, spleen cytotoxic precursors, or clearance of virus. However, Y100F-Ig treatment had a clear effect on lung effector cell function. Secretion of IFN-γ by bronchoalveolar lavage fluid CD8+ T lymphocytes in vitro was decreased, and the number of virus-specific CD8+ T lymphocytes in the bronchoalveolar lavage fluid and lungs of infected mice was reduced. These results indicate that CD28-dependent costimulation is important in the antiviral immune response to an influenza virus infection. The individual CD28 ligand, CD80, is important for some lung immune responses and cannot always be compensated for by CD86.
Bovine and Human Insulin Activate CD8+-Autoreactive CTL Expressing Both Type 1 and Type 2 Cytokines in C57BL/6 Mice
Authors: Ma Hakling , Ke Yong , Li Qingqin , Kapp Judith A.
Published: April 2014

CD8+ T cells down-regulate a variety of immune responses. For example, porcine and human insulin do not stimulate Abs in C57BL/6 mice because CD8+ T cells inhibit CD4+ helper T cells. By contrast, bovine insulin induces Ab in C57BL/6 mice, and removal of CD8+ T cells does not alter this response. This raises the question of whether porcine, but not bovine, insulin activates CD8+ T cells or whether both insulins activate CD8+ T cells but CD4+ helper T cells are differentially inhibited by them. In this study, we show that insulin-specific CD8+ CTL can be cultured from C57BL/6 mice primed with either bovine or human insulin in CFA. Thus, exogenous Ags, besides OVA, induce CD8+ CTL when administered in an adjuvant, suggesting this is a typical response. These CTL are H-2Kb restricted and produce IL-5, IL-10, IFN-γ, and small amounts of IL-4, which is distinct from IFN-γ and TNF-α that are typically secreted by virus-specific CTL. Moreover, the CTL primed with either bovine or human insulin recognize an A-chain peptide that is identical to the mouse insulin sequence. That foreign proteins, which are closely related to self-proteins, activated autoreactive, CD8+ T cells in vivo is a novel finding. It raises the possibility that self-reactive CTL may be activated by cross-reacting Ags and once activated they might participate in autoimmunity. These results also suggest that down-regulation of insulin-specific responses by autoreactive CD8+ T cells is most likely due to the differential sensitivity of bovine and human insulin-specific CD4+ T cells.
Cutting Edge: Persistent Fetal Microchimerism in T Lymphocytes Is Associated with HLA-DQA1*0501: Implications in Autoimmunity
Authors: Lambert Nathalie C. , Evans Paul C. , Hashizumi Tanya L. , Maloney Sean , Gooley Ted , Furst Dan E. , Nelson J. Lee
Published: April 2014

The host’s MHC genotype plays a critical role in susceptibility to autoimmune diseases. We previously proposed that persistent fetal microchimerism from pregnancy contributes to the pathogenesis of autoimmune diseases such as scleroderma. In the current study, we investigated whether the specific host MHC genotype is associated with persistent microchimerism among T lymphocytes in women with scleroderma and in healthy women. Fetal microchimerism among T lymphocytes was strongly associated with HLA DQA1*0501 of the mother (odds ratio (OR) = 13.5, p = 0.007, p corrected (pc) = 0.06) and even more strongly with DQA1*0501 of the son (OR = ∞; p = 0.00002, pc = 0.0002). This is the first description of an association between persistent fetal microchimerism in maternal T lymphocytes and specific HLA class II alleles. Although the association was observed in both healthy women and in women with scleroderma, the finding suggests an additional route by which HLA genes might contribute to susceptibility to autoimmune disease.
Regulation of Murine Airway Eosinophilia and Th2 Cells by Antigen-Conjugated CpG Oligodeoxynucleotides as a Novel Antigen-Specific Immunomodulator
Authors: Shirota Hidekazu , Sano Kunio , Kikuchi Tadashi , Tamura Gen , Shirato Kunio
Published: April 2014

The characteristic features of bronchial asthma reflect the orchestrated activity of Th2 cells. Oligodeoxynucleotides containing CpG motifs (CpG) have recently been highlighted as an immunomodulator that biases toward a Th1-dominant phenotype. We have previously reported that intratracheal coadministration of CpG and allergen inhibited airway eosinophilia and hyperresponsiveness in a synergistic manner. To substantiate the synergism between CpG and Ag, we introduced a covalently linked conjugate between CpG and Ag and examined the efficacy on airway eosinophilia and Th2 cytokine production. We found that the conjugated form of CpG plus Ag was 100-fold more efficient in regulating airway eosinophilia than the unconjugated mixture. The inhibitory effects lasted for at least 2 mo. The inhibition of airway eosinophilia by the conjugate was Ag specific and associated with an improvement of the airway hyperresponsiveness and the unresponsiveness of the Ag-specific Th2 cells in the regional lymph nodes. The CpG-Ag conjugate was 100-fold more effective than the unconjugated mixture for inducing in vitro Th1 differentiation in an IL-12-dependent manner. Our data show that CpG conjugated to Ag can work as a novel Ag-specific immunomodulator and imply that inhalation of allergen-CpG conjugate could be a desensitization therapy for patients with bronchial asthma.
A Soluble Lymphocyte Activation Gene-3 Molecule Used as a Vaccine Adjuvant Elicits Greater Humoral and Cellular Immune Responses to Both Particulate and Soluble Antigens
Authors: El mir Samir , Triebel Frédéric
Published: April 2014

The lymphocyte activation gene-3 (LAG-3) product is a MHC class II ligand that has been used in vivo to stimulate MHC class II+ APCs to increase tumor-specific immune responses. We investigated whether LAG-3 could also play an adjuvant role in vivo for the induction of humoral and CD4 or CD8 cell-mediated immune responses when immunizing mice with a particulate (hepatitis B surface Ag) or soluble (OVA) Ag. In both cases, coadministration of 1 μg of a soluble fusion protein between murine LAG-3 and the Fc fraction of a murine IgG2a mAb (mLAG-3Ig) as a vaccine adjuvant induced or increased CTL responses to the corresponding MHC class I-restricted peptide. In addition, splenocytes of mice vaccinated with either the particulate or soluble Ag plus mLAG-3Ig exhibited a significantly greater proliferative response than did splenocytes of mice immunized with Ag and a control Ig molecule. Similarly, these splenocytes had a greater Th1- but not Th2-type cytokine response. Finally, mice immunized with Ag plus mLAG-3Ig produced higher titers of Abs than mice immunized with Ag and a control Ig molecule. Thus, these data provide evidence of a novel means of improving the immunogenicity of subunit vaccines.
B Cell Responses to a Peptide Epitope. IX. The Kinetics of Antigen Binding Differentially Regulates Costimulatory Capacity of Activated B Cells
Authors: Vijayakrishnan Lalitha , Natarajan Krishnamurthy , Manivel Venkatasamy , Raisuddin Sheikh , Rao Kanury V. S.
Published: April 2014

We explore the possible mechanism by which association rates of Ag with activated B cells influences the ability of the latter to selectively recruit Th subsets. Our system used cocultures of Ag-activated B and T cells, where the Ag was a synthetic peptide, G41CT3. Restimulation was with either peptide G41CT3 or its analogue, G28CT3. Peptide G28CT3 has been previously shown to display a higher on rate, relative to the homologous peptide G41CT3, of binding to G41CT3-activated B cells. This difference in on rates was eventually exerted at the level of IFN-γ, but not of IL-10, induction from T cells, with peptide G28CT3 proving more effective. However, various treatment regimens rendered peptide G41CT3 as potent as peptide G28CT3 at eliciting IFN-γ responses from the above cultures. This included simultaneous treatment of B cells with peptide G41CT3 and the protein tyrosine kinase inhibitor tyrphostin. Alternatively, pretreatment of B cells with a peptide representing only the B cell epitope constituent of peptide G28CT3 (G28) was also equally effective. Subsequent experiments revealed that IFN-γ production from activated T cells resulted from an engagement of CD28 by B7-1 on the B cell surface. Finally, the extent of cell surface B7-1 up-regulation on activated B cells was dependent on the on rate of Ag binding to the membrane-bound Ig receptor. Thus, cumulative results suggest that the kinetics of Ag binding to activated B cells can differentially regulate intracellular signaling. This influences selective costimulatory molecule expression, with its consequent effects on relative Th subset activation.
CD4+ T Cell Responses to Self- and Mutated p53 Determinants During Tumorigenesis in Mice
Authors: Fedoseyeva Eugenia V. , Boisgérault Florence , Anosova Natalie G. , Wollish Wendy S. , Arlotta Paola , Jensen Peter E. , Ono Santa J. , Benichou Gilles
Published: April 2014

We analyzed CD4+ T helper responses to wild-type (wt) and mutated (mut) p53 protein in normal and tumor-bearing mice. In normal mice, we observed that although some self-p53 determinants induced negative selection of p53-reactive CD4+ T cells, other p53 determinants (cryptic) were immunogenic. Next, BALB/c mice were inoculated with J774 syngeneic tumor cell line expressing mut p53. BALB/c tumor-bearing mice mounted potent CD4+ T cell responses to two formerly cryptic peptides on self-p53. This response was characterized by massive production of IL-5, a Th2-type lymphokine. Interestingly, we found that T cell response was induced by different p53 peptides depending upon the stage of cancer. Mut p53 gene was shown to contain a single mutation resulting in the substitution of a tyrosine by a histidine at position 231 of the protein. Two peptides corresponding to wt and mutated sequences of this region were synthesized. Both peptides bound to the MHC class II-presenting molecule (Ed) with similar affinities. However, only mut p53.225–239 induced T cell responses in normal BALB/c mice, a result strongly suggesting that high-affinity wt p53.225–239 autoreactive T cells had been eliminated in these mice. Surprisingly, CD4+ T cell responses to both mut and wt p53.225–239 peptides were recorded in J774 tumor-bearing mice, a phenomenon attributed to the recruitment of low-avidity p53.225–239 self-reactive T cells.
Down-Regulation of the Orphan Nuclear Receptor RORγt Is Essential for T Lymphocyte Maturation
Authors: He You-Wen , Beers Courtney , Deftos Michael L. , Ojala Ethan W. , Forbush Katherine A. , Bevan Michael J.
Published: April 2014

Thymocyte development is a tightly regulated process. CD4+CD8+ double-positive (DP) immature thymocytes exhibit distinct phenotypic features from mature T cells; they express only 10% of surface TCR that are found on mature T cells and do not proliferate and produce IL-2 in response to stimulation. In this report we show that transgenic expression of the orphan nuclear receptor RORγt in mature T cells down-regulates their surface TCR expression. The RORγt transgene inhibits IL-2 production by mature T cells, and this inhibition may be partially due to the inhibitory effect of RORγt on c-Rel transcription. Furthermore, ectopic expression of RORγt inhibits the proliferation of mature and immature T cells. These results, together with its predominant expression in DP thymocytes, suggest that RORγt controls these distinct phenotypic features of DP thymocytes. Our data suggest that down-regulation of RORγt expression in thymocytes is essential for their maturation.
Ligand-Specific Selection of MHC Class II-Restricted Thymocytes in Fetal Thymic Organ Culture
Authors: Kersh Gilbert J. , Engle Deborah L. , Williams Calvin B. , Allen Paul M.
Published: April 2014

Positive and negative selection of thymocytes is determined by the specificity of the TCR and signaling through its associated molecules. We have studied selection of thymocytes bearing a MHC class II-restricted TCR using fetal thymic organ culture. This system allows the addition of peptides to the already diverse panoply of endogenous peptide ligands and is useful for analyzing ligand-specific negative selection of CD4 single positive (CD4SP) thymocytes. The data reveal that the ability of a given ligand to mediate negative selection is related to its dissociation rate from the TCR. We find that negative selection is very sensitive, and only the weakest ligand that we can identify fails to induce negative selection. None of the numerous peptides tested were able to induce an increase in CD4SP thymocytes. In addition, the ligands that induce negative selection of CD4SP thymocytes also cause an increase in numbers of CD8SP thymocytes bearing high levels of the class II-restricted TCR. Although these cells have a cell surface phenotype consistent with positive selection, they most likely represent cells in the process of negative selection. Further analysis reveals that these cells are not induced by these ligands in intact adult animals and that their induction is probably only revealed in the organ culture system.
Cooperation Among Stat1, Glucocorticoid Receptor, and PU.1 in Transcriptional Activation of the High-Affinity Fcγ Receptor I in Monocytes
Authors: Aittomäki Saara , Pesu Marko , Groner Bernd , Jänne Olli A. , Palvimo Jorma J. , Silvennoinen Olli
Published: April 2014

IFN-γ and glucocorticoids regulate inflammatory and immune responses through Stat1 and glucocorticoid receptor (GR) transcription factors, respectively. The biological responses to these polypeptides are determined by integration of various signaling pathways in a cell-type and promoter-dependent manner. In this study we have characterized the molecular basis for the functional cooperation between IFN-γ and dexamethasone (Dex) in the induction of the high-affinity Fcγ receptor I (FcγRI) in monocytes. Dex did not affect IFN-γ-induced Stat1 DNA binding activity or induce novel DNA-binding complexes to the FcγRI promoter. By using cell systems lacking functional GR or Stat1, we showed that GR stimulated Stat1-dependent transcription in a ligand-dependent manner, while Stat1 did not influence GR-dependent transcription. The cooperation required phosphorylation of Tyr701, DNA binding, and the trans-activation domain of Stat1, but did not involve Ser727 phosphorylation of Stat1 or physical interaction between GR and Stat1. The costimulatory effect of Dex was not dependent on a consensus glucocorticoid response element in the Stat1-responsive promoters, but required the DNA-binding and trans-activation functions of GR, and Dex-induced protein synthesis. GR activated the natural FcγRI promoter construct, and this response required both Stat1 and the Ets family transcription factor PU.1. Previously, physical association between GR and Stat5 has been shown to enhance Stat5-dependent and suppress GR-dependent transcription. The results shown here demonstrate a distinct, indirect mechanism of cross-modulation between cytokine and steroid receptor signaling that integrates Stat1 and GR pathways with cell type-specific PU.1 transcription factor in the regulation of FcγRI gene transcription.
Antigenic Epitopes Regulate the Phenotype of CD8+ CTL Primed by Exogenous Antigens
Authors: Ma Hakling , Kapp Judith A.
Published: April 2014

We previously reported that insulin-specific, MHC class I-restricted CTL precursors can be primed by injecting C57BL/6 mice with bovine insulin in CFA. These bovine insulin-primed CTL displayed a type 0 CTL phenotype, producing IL-4, IL-5, IL-10, low levels of IFN-γ, but no TNF-α. By contrast, CTL generated from C57BL/6 mice primed with OVA in CFA produced IFN-γ and TNF-α but no IL-4, IL-5, or IL-10 and therefore were classified as type 1 CTL. Although CD4+ T cell subsets have been compared extensively in the literature, CTL subsets are less well characterized. Here, the phenotype, function, and requirements for the in vivo activation of type 1 and type 0 CTL cells were studied. Although both types of CTL express many of the same cell-surface Ags, OVA-specific CTL but not bovine insulin-primed CTL expressed CT-1, a carbohydrate epitope of CD45, and bovine insulin-primed CTL but not OVA-specific CTL expressed Fas constitutively. Priming of CTL was abrogated by depletion of phagocytic cells but not CD4+ T cells, whereas depletion of CD4+ T cells but not phagocytic cells inhibited Ab responses in the same mice. Neither endogenous IL-4 nor the dose of priming Ag altered the CTL phenotypes, but the antigenic peptides of OVA and bovine insulin were key to determining the differentiation of either type 1 or type 0 CTL. To our knowledge, this is the first time that antigenic epitopes have been demonstrated to influence the phenotype of Ag-specific CTL responses. These results may be relevant to the development of peptide vaccines in which a particular type of CTL response is desired.
FTY720 Immunosuppression Impairs Effector T Cell Peripheral Homing Without Affecting Induction, Expansion, and Memory
Authors: Pinschewer Daniel D. , Ochsenbein Adrian F. , Odermatt Bernhard , Brinkmann Volker , Hengartner Hans , Zinkernagel Rolf M.
Published: April 2014

FTY720 (2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride) prolongs survival of solid organ allografts in animal models. Mechanisms of FTY720 immunomodulation were studied in mice infected with lymphocytic choriomeningitis virus (LCMV) to assess T cell responses or with vesicular stomatitis virus to evaluate Ab responses. Oral FTY720 (0.3 mg/kg/day) did not affect LCMV replication and specific CTL and B cells were induced and expanded normally. Moreover, the anti-viral humoral immune responses were normal. However, FTY720 treatment showed first a shift of overall distribution of CTL from the spleen to peripheral lymph nodes and lymphocytopenia was observed. This effect was reversible within 7–21 days. Together with unimpaired T and B cell memory after FTY720 treatment, this finding rendered enhancement of lymphocyte apoptosis by FTY720 in vivo unlikely. Secondly, the delayed-type hypersensitivity reaction to a viral MHC class I-presented peptide was markedly reduced by FTY720. These results were supported by impaired circulation of LCMV specific TCR transgenic effector lymphocytes in the peripheral blood and reduced numbers of tissue infiltrating CTL in response to delayed-type hypersensitivity reaction. Thirdly, in a CD8+ T cell-mediated diabetes model in a transgenic mouse expressing the LCMV glycoprotein in the islets of the pancreas, FTY720 delayed or prevented disease by reducing islet-infiltrating CTL. Thus, FTY720 effectively reduced recirculation of CD8+ effector T cells and their recruitment to peripheral lesions without affecting the induction and expansion of immune responses in secondary lymphoid organs. These properties may offer the potential to treat ongoing organ-specific T cell-mediated immunopathologic disease.
Size Estimate of the αβ TCR Repertoire of Naive Mouse Splenocytes
Authors: Casrouge Armanda , Beaudoing Emmanuel , Dalle Sophie , Pannetier Christophe , Kanellopoulos Jean , Kourilsky Philippe
Published: April 2014

The diversity of the T cell repertoire of mature T splenocytes is generated, in the thymus, by pairing of α and β variable domains of the αβ TCR and by the rearrangements of various gene segments encoding these domains. In the periphery, it results from competition between various T cell subpopulations including recent thymic migrants and long-lived T cells. Quantitative data on the actual size of the T cell repertoire are lacking. Using PCR methods and extensive sequencing, we have measured for the first time the size of the TCR-αβ repertoire of naive mouse T splenocytes. There are 5–8 × 105 different nucleotide sequences of BV chains in the whole spleen of young adult mice. We have also determined the size of the BV repertoire in a subpopulation of AV2+ T splenocytes, which allows us to provide a minimum estimate of the αβ repertoire. We find that the mouse spleen harbors about 2 × 106 clones of about 10 cells each. This figure, although orders of magnitude smaller than the maximum theoretical diversity (estimated up to 1015), is still large enough to maintain a high functional diversity.
Vesnarinone Suppresses TNF-Induced Activation of NF-κB, c-Jun Kinase, and Apoptosis
Authors: Manna Sunil K. , Aggarwal Bharat B.
Published: April 2014

Vesnarinone, a synthetic quinolinone derivative used in the treatment of cardiac failure, exhibits immunomodulatory, anti-inflammatory, and cell growth regulatory properties. The mechanisms underlying these properties are not understood, but due to the critical role of nuclear transcription factor NF-κB in these responses, we hypothesized that vesnarinone must modulate NF-κB activation. We investigated the effect of vesnarinone on NF-κB activation induced by inflammatory agents. Vesnarinone blocked TNF-induced activation of NF-κB in a concentration- and time-dependent manner. This effect was mediated through inhibition of phosphorylation and degradation of IκBα, an inhibitor of NF-κB. The effects of vesnarinone were not cell type specific, as it blocked TNF-induced NF-κB activation in a variety of cells. NF-κB-dependent reporter gene transcription activated by TNF was also suppressed by vesnarinone. The TNF-induced NF-κB activation cascade involving TNF receptor 1-TNF receptor associated death domain-TNF receptor associated factor 2 NF-κB-inducing kinase-IKK was interrupted at the TNF receptor associated factor 2 and NF-κB-inducing kinase sites by vesnarinone, thus suppressing NF-κB reporter gene expression. Vesnarinone also blocked NF-κB activation induced by several other inflammatory agents, inhibited the TNF-induced activation of transcription factor AP-1, and suppressed the TNF-induced activation of c-Jun N-terminal kinase and mitogen-activated protein kinase kinase. TNF-induced cytotoxicity, caspase activation, and lipid peroxidation were also abolished by vesnarinone. Overall, our results indicate that vesnarinone inhibits activation of NF-κB and AP-1 and their associated kinases. This may provide a molecular basis for vesnarinone’s ability to suppress inflammation, immunomodulation, and growth regulation.
IFN Regulatory Factor-1-Mediated Transcriptional Activation of Mouse STAT-Induced STAT Inhibitor-1 Gene Promoter by IFN-γ
Authors: Saito Hiroshi , Morita Yoshiaki , Fujimoto Minoru , Narazaki Masashi , Naka Tetsuji , Kishimoto Tadamitsu
Published: April 2014

STAT-induced STAT inhibitor-1 (SSI-1), also referred to as suppressor of cytokine signaling-1 and JAK-binding protein, is a member of a new family, the members of which are negative regulators of cytokine signals. SSI-1 is induced by various cytokines; however, the transcriptional mechanism of the SSI-1 gene is not fully understood. Here, we showed that transcription of the mouse SSI-1 gene was initiated from six adjoining sites accompanying three GC boxes and a single GC box-like element near them, but not from the TATA box or an initiator sequence. We also showed that IFN-γ induced SSI-1 mRNA more strongly than IL-6 in NIH-3T3 fibroblasts and that this IFN-γ effect was mediated by Stat1. To determine the signal pathway downstream of Stat1, transcriptional activities of several mutant promoters were examined. The region mediating stimulatory effect of IFN-γ to the gene transcription was localized to the −88/−60 region containing three tandem GAAA units, named variant IFN-γ-responsive element (VIRE), while four IFN-γ activation site (GAS)-like elements located far upstream were not related to the IFN-γ response. Gel-shift assays revealed that IFN-γ induced IFN regulatory factor-1 (IRF-1) binding to VIRE, but not that of IRF-2 or three components of ISGF3. Furthermore, forced expression of IRF-1 mimicked and that of IRF-2 inhibited the stimulatory effect of IFN-γ on SSI-1 gene transcription. Finally, mouse embryonal fibroblasts lacking IRF-1 showed impaired SSI-1 mRNA induction by IFN-γ. These results demonstrated that IRF-1, which is induced by activation of Stat1, mediated transcriptional activation of the SSI-1 gene by IFN-γ via VIRE.