bluuebunny/crossref_metadata_2025 huggingface dataset:
Disparate Intracellular Processing of Human IL-12 Preprotein Subunits: Atypical Processing of the P35 Signal Peptide
Authors: Murphy Finbarr J. , Hayes Mark P. , Burd Parris R.
Published: April 2014

IL-12 is a heterodimeric cytokine produced by APC that critically regulates cell-mediated immunity. Because of its crucial function during immune responses, IL-12 production is stringently regulated, in part through transcriptional control of its p35 subunit, which requires the differentiative effects of IFN-γ for expression. To determine whether post-transcriptional aspects of IL-12 production might be regulated, we examined intracellular protein processing of each subunit. We report here that p40 and p35 subunits are processed by disparate pathways. Whereas processing of p40 conforms to the cotranslational model of signal peptide removal concomitant with translocation into the endoplasmic reticulum (ER), processing of p35 does not. Translocation of the p35 preprotein into the ER was not accompanied by cleavage of the signal peptide; rather, removal of the p35 signal peptide occurred via two sequential cleavages. The first cleavage took place within the ER, and the cleavage site localized to the middle of the hydrophobic region of the signal peptide. Although the preprotein was glycosylated upon entry into the ER, its glycosylation status did not affect primary cleavage. Subsequently, the remaining portion of the p35 signal peptide was removed by a second cleavage, possibly involving a metalloprotease, concomitant with additional glycosylation and secretion. Secretion could be inhibited by mutation of the second cleavage site or by inhibition of glycosylation with tunicamycin. In contrast, p40 secretion was not affected by inhibition of glycosylation. Our findings demonstrate that IL-12 subunits are processed by disparate pathways and suggest new modalities for regulation of IL-12 production.
The Cytoplasmic Tyrosine Motifs in Full-Length Glycoprotein 130 Have Different Roles in IL-6 Signal Transduction
Authors: Schmitz Jochen , Dahmen Heike , Grimm Carsten , Gendo Cornelia , Müller-Newen Gerhard , Heinrich Peter C. , Schaper Fred
Published: April 2014

The function of the signal-transducing receptor subunit glycoprotein 130 (gp130) in the IL-6-receptor complex has previously been studied using carboxyl-terminal deletion mutants or a truncated molecule of ∼60 membrane-proximal amino acids (containing box 1 and box 2) linked to the individual gp130 tyrosine motifs. However, the redundancy of the tyrosine motifs within the cytoplasmic part of gp130 has been neglected. Here we describe the analysis of the function of the individual cytoplasmic tyrosine residues of gp130 in the context of the full-length receptor protein in IL-6 signaling as measured by STAT activation, acute phase protein induction, and stimulation of proliferation. Add-back receptor mutants containing only one cytoplasmic tyrosine have been generated and tested for their efficiency in IL-6 signal transduction. Our studies revealed that tyrosine motifs which have been described to recruit STAT proteins are not equivalent with respect to their potential to activate STAT factors and acute phase protein gene promoters: the two distal tyrosines, Tyr905 and Tyr915, of gp130 were more potent than Tyr767 and Tyr814. Surprisingly, Tyr905 and Tyr915 mediate acute phase protein gene promoter activation stronger than the wild-type receptor containing all six cytoplasmic tyrosine residues. In contrast, Ba/F3 cells stably transfected with add-back receptors containing Tyr767 or Tyr905 were more sensitive to IL-6-induced proliferation than cells expressing the other add-back receptor mutants. Thus, the tyrosine residues in the cytoplasmic part of gp130 were found to contribute differentially to IL-6 signal transduction in the full- length gp130 protein.
Involvement ofmi-Transcription Factor in Expression of α-Melanocyte-Stimulating Hormone Receptor in Cultured Mast Cells of Mice
Authors: Adachi Shiro , Morii Eiichi , Kim Dae-ki , Ogihara Hideki , Jippo Tomoko , Ito Akihiko , Lee Young-Mi , Kitamura Yukihiko
Published: April 2014

The microphthalmia (mi) locus encodes a member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of transcription factors (MITF). We have reported that expression of several genes was impaired in cultured mast cells (CMCs) of mi/mi mice due to a defective transactivation ability of mutant MITF (mi-MITF). We also found that mi/mi CMCs did not express a receptor (MC1R) for α-melanocyte-stimulating hormone. The overexpression of the wild-type (+/+) MITF but not mi-MITF normalized the expression of the MC1R in mi/mi CMCs, indicating the involvement of +-MITF in the MC1R gene expression. Next, we analyzed the promoter region of the MC1R gene by the transient cotransfection assay. The luciferase construct under the control of the MC1R promoter and the cDNA-encoding +-MITF or mi-MITF were cotransfected into NIH/3T3 fibroblasts. The cotransfection of +-MITF but not mi-MITF increased the luciferase activity. There were five CANNTG motifs recognized by bHLH-Zip-type transcription factors in the cloned promoter region. We found +-MITF bound two of five CANNTG motifs, and both motifs were essential for the transactivation of the MC1R gene by +-MITF. These results indicated that +-MITF directly transactivated the MC1R gene through these two motifs.
Contribution of Individual Amino Acids Within MHC Molecule or Antigenic Peptide to TCR Ligand Potency
Authors: Hemmer Bernhard , Pinilla Clemencia , Gran Bruno , Vergelli Marco , Ling Nick , Conlon Paul , McFarland Henry F. , Houghten Richard , Martin Roland
Published: April 2014

The TCR recognition of peptides bound to MHC class II molecules is highly flexible in some T cells. Although progress has been made in understanding the interactions within the trimolecular complex, to what extent the individual components and their amino acid composition contribute to ligand recognition by individual T cells is not completely understood. We investigated how single amino acid residues influence Ag recognition of T cells by combining several experimental approaches. We defined TCR motifs for CD4+ T cells using peptide synthetic combinatorial libraries in the positional scanning format (PS-SCL) and single amino acid-modified peptide analogues. The similarity of the TCR motifs defined by both methods and the identification of stimulatory antigenic peptides by the PS-SCL approach argue for a contribution of each amino acid residue to the overall potency of the antigenic peptide ligand. In some instances, however, motifs are formed by adjacent amino acids, and their combined influence is superimposed on the overall contribution of each amino acid within the peptide epitope. In contrast to the flexibility of the TCR to interact with different peptides, recognition was very sensitive toward modifications of the MHC-restriction element. Exchanges of just one amino acid of the MHC molecule drastically reduced the number of peptides recognized. The results indicate that a specific MHC molecule not only selects certain peptides, but also is crucial for setting an affinity threshold for TCR recognition, which determines the flexibility in peptide recognition for a given TCR.
TNF-α Regulates Transforming Growth Factor-α Expression in Regenerating Murine Liver and Isolated Hepatocytes
Authors: Gallucci Randle M. , Simeonova Petia P. , Toriumi Wataru , Luster Michael I.
Published: April 2014

TNF-α is a pleotropic proinflammatory cytokine that has been implicated as a contributing factor in a number of disease processes, primarily through its ability to induce the expression of inflammatory and cytotoxic mediators. TNF-α is also involved in cell growth accompanying the healing process in multiple organ systems and influences liver repair following hepatotoxic damage or regeneration following partial hepatectomy. In this respect, TNF-α is a known mitogen for hepatocytes. In this paper we describe a novel role for TNF-α in the modulation of expression of TGF-α, the latter being a complete hepatocyte mitogen. TNF-α directly up-regulates TGF-α mRNA by up to 7-fold in isolated mouse hepatocytes, whereas neutralization of TNF-α significantly decreased liver mRNA and protein expression of TGF-α following chemical-induced hepatotoxicity. That TNF-α directly stimulated TGF-α was suggested by the inability of either anti-IL-6 Abs or cycloheximide to inhibit TNF-α-induced TGF-α expression in hepatocytes. However, in the presence of anti-TGF-α neutralizing Abs, the mitogenic activity of TNF-α is abrogated. Using cells transfected with the TGF-α promoter, and an RNA polymerase inhibitor, it was shown that TNF-α modulates TGF-α expression through both pre- and posttranscriptional events. Taken together, these data suggest that TNF-α participates in liver repair and regeneration, in part, by directly inducing the expression of TGF-α.
Evidence for a Stepwise Evolution of the CD3 Family
Authors: Göbel Thomas W. F. , Dangy Jean-Pierre
Published: April 2014

The three CD3 components of the TCR complex are encoded as clustered genes in mammals. The evolution of such a multimeric complex is likely to occur stepwise. The chicken CD3 cluster was entirely sequenced, and, in contrast to mammals, only two chicken CD3 genes were found to be physically linked to the unrelated genes HZW10 and epithelial V-like Ag flanking both sides of the CD3 cluster. Biochemical analyses of CD3 immunoprecipitates confirmed the presence of only two CD3 proteins and revealed an essential role for CD3γδ glycosylation during assembly. Functional analyses indicated that the chicken TCR/CD3 complex was efficiently down-regulated by phorbol ester treatment, demonstrating the integrity of a CD3γ-like cytoplasmic internalization motif. These data argue for a stepwise CD3 evolution, with major differences in the TCR/CD3 structure between mammalian and nonmammalian vertebrates setting a basis for the understanding of the CD3 phylogeny and proving the ancestral nature of the CD3γδ protein.
Transgenic Expression of Ly-49A in Thymocytes Alters Repertoire Selection
Authors: Pauza Mary , Smith Kathleen M. , Neal Heather , Reilly Christina , Lanier Lewis L. , Lo David
Published: April 2014

A T cell-specific Ly-49A transgene inhibits TCR-mediated activation in the presence of H-2Dd. Expression of this transgene by developing thymocytes impairs negative selection evidenced by a failure to delete potentially autoreactive T cells and development of a graft-vs-host-disease-like syndrome. In mice carrying both the Ly-49A and a class II-restricted TCR transgene, positive selection was lost, but only when H-2Dd was present on thymic epithelium. These results are consistent with models suggesting that thymic selection is dependent on the perceived intensity of TCR signaling. More interestingly, these results show that Ly-49A does not simply provide a strict on/off switch for T cell responses. Since Ly-49A may shift the signaling threshold of TCR-induced triggering, inducible expression of Ly-49A may regulate peripheral memory/activated T cells by raising the threshold for T cell reactivation.
Frequency and Characterization of Phenotypic Ig Heavy Chain Allelically Included IgM-Expressing B Cells in Mice
Authors: Barreto Vasco , Cumano Ana
Published: April 2014

Ig H chain (IgH) allelic exclusion remains a puzzling topic. Here, we address the following question: Do phenotypic IgH allelically included cells exist in normal mice and, if so, at what frequency? Sorted cells from heterozygous mice were evaluated for the expression of both IgM allotypes by double intracytoplasmic stainings. Dual expressors were found at a frequency of 1 in 104 splenic B cells. These data were confirmed by direct sequencing of IgH-rearranged alleles obtained after single cell (or clone) PCR on dual expressors. Typically, these cells have one rearranged J558 VH whereas, in the other allele, a D-proximal VH gene is used. Interestingly, dual expressors have rearranged IgH alleles with similar CDR3 lengths. These results show that, in contrast to the κ L chain and the TCR β-chain, IgH allelic exclusion is the result of an extremely stringent mechanism. We discuss two non-mutually exclusive scenarios for the origin of IgH dual expressors: 1) IgH allelically included cells arise when the first allele to rearrange productively is unable to form a pre-BCR; dual expressors could be a subset of this population in which, upon conventional L chain rearrangement, both IgH are expressed at the surface; and 2) synchronous rearrangement of the IgH alleles.
Extension of HLA-A*0201-Restricted Minimal Epitope by Nε-Palmitoyl-lysine Increases the Life Span of Functional Presentation to Cytotoxic T Cells
Authors: Loing Estelle , Andrieu Muriel , Thiam Kader , Schörner Dominik , Wiesmüller Karl-Heinz , Hosmalin Anne , Jung Günther , Gras-Masse Hélène
Published: April 2014

The delineation of the minimal requirements for efficient delivery of functional cytotoxic epitopes into APC could be a step toward the definition of “minimal length” lipopeptides for the modulation of CTL activity. Several analogues of the HLA-A*0201-restricted HIV-1 polymerase (pol476–484) minimal cytotoxic epitope were obtained by modifying P0, P1, or P10 positions by a single Nε-palmitoyl-lysine residue. The use of fluorescent derivatives confirmed the cell-permeating activities and suggested that a P0- and a P1-modified lipopeptide possessing ionizable extremities fulfills the structural requirements for MHC loading. The expressions of HLA-peptide complexes at the surface of TAP-deficient cells incubated with the parent epitope or lipopeptide derivatives were compared, in terms of intensity and stability. Both lipopeptides induced a considerably prolonged expression of conformationally correct complexes, which were dependent on the integrity of the exocytosis pathway, suggesting a dynamic mechanism of formation or reloading of the complexes from an intracellular pool. The agonistic activities of the different HLA-peptide complexes were evaluated using two independent T cell lines from HIV-infected donors. We report that a lipodecapeptide obtained by N-terminal addition of a Nε-palmitoyl-lysine to the pol476–484 epitope was able to increase the life span of functional presentation to cytotoxic T cells specific for the parent peptide.
Bacterial Clearance and Survival Are Dependent on CXC Chemokine Receptor-2 Ligands in a Murine Model of Pulmonary Nocardia asteroides Infection
Authors: Moore Thomas A. , Newstead Michael W. , Strieter Robert M. , Mehrad Borna , Beaman Blaine L. , Standiford Theodore J.
Published: April 2014

Survival from murine pulmonary nocardiosis is highly dependent on CXC chemokine receptor-2 (CXCR2) ligand-mediated neutrophil chemotaxis and subsequent clearance of the infectious agent Nocardia asteroides. Intratracheal inoculation of N. asteroides rapidly up-regulated the CXC chemokines macrophage inflammatory protein-2 (MIP-2) and KC within 24 h, with levels remaining elevated through day 3 before returning to near baseline levels by day 7. Coinciding with elevated MIP-2 and KC were the rapid recruitment of neutrophils and clearance of the organism. Anti-Ly-6G Ab-mediated neutrophil depletion before bacterial challenge resulted in strikingly increased mortality to N. asteroides infection. The relative contribution of MIP-2 in neutrophil recruitment was examined by anti-MIP-2 Ab treatment before nocardial infection. MIP-2 neutralization had no detrimental effects on survival, neutrophil recruitment, or bacterial clearance, suggesting the usage of additional or alternative CXCR2-binding ligands. The importance of the CXC family of chemokines was determined by the administration of an anti-CXCR2 Ab capable of blocking ligand binding in vivo. Anti-CXCR2 treatment greatly increased mortality by preventing neutrophil migration into the lung. Paralleling this impaired neutrophil recruitment was a 100-fold increase in lung bacterial burden. Combined, these observations indicate a critical role for neutrophils and CXC chemokines during nocardial pneumonia. These data directly link CXCR2 ligands and neutrophil recruitment and lend further support to the concept of CXC chemokine redundancy. For infections highly dependent on neutrophils, such as nocardial pneumonia, this is of critical importance.
Type 1 and Type 2 CD8+ Effector T Cell Subpopulations Promote Long-Term Tumor Immunity and Protection to Progressively Growing Tumor
Authors: Dobrzanski Mark J. , Reome Joyce B. , Dutton Richard W.
Published: April 2014

Cytolytic CD8+ effector cells fall into two subpopulations based on cytokine secretion. Type 1 CD8+ T cells (Tc1) secrete IFN-γ, whereas type 2 CD8+ T cells (Tc2) secrete IL-4, IL-5, and IL-10. Using an OVA-transfected B16 lung metastases model, we assessed the therapeutic effects of adoptively transferred OVA-specific Tc1 and Tc2 subpopulations in mice bearing established pulmonary malignancy. Effector cell-treated mice exhibiting high (5 × 105) tumor burdens experienced significant (p < 0.05) delays in mortality compared with those of untreated control mice, whereas high proportions (70–90%) of mice receiving therapy with low (1 × 105) tumor burdens survived indefinitely. Long-term tumor immunity was evident by resistance to lethal tumor rechallenge, heightened levels of systemic OVA Ag-specific CTL responses ex vivo, and detection of long-lived TCR transgene-positive donor cells accompanied by an elevation in the total numbers of CD8+ CD44high activated and/or memory T cells at sites of tumor growth. Long-lasting protection by Tc2 and Tc1 effector cells were dependent, in part, on both the level of tumor burden and effector cell-derived IL-4, IL-5, and IFN-γ, respectively. We conclude that Tc1 and Tc2 effector cells provide immunity by different mechanisms that subsequently potentiate host-derived antitumor responses.
Decreased Cytomegalovirus Expression Following Proinflammatory Cytokine Treatment of Primary Human Astrocytes
Authors: Cheeran Maxim C.-J. , Hu Shuxian , Gekker Genya , Lokensgard James R.
Published: April 2014

Understanding the influence of immune effector mechanisms on CMV infection of the CNS may facilitate the development of immunotherapies for viral encephalitis. Using cultures of highly purified, fully permissive primary human astrocytes, proinflammatory cytokines, but not antiinflammatory cytokines or β-chemokines, were found to inhibit CMV expression, DNA synthesis, and replication. Treatment with certain proinflammatory cytokines 24 h before CMV infection markedly suppressed viral expression in astrocytes. TNF-α, IL-1β, and IFN-γ all inhibited CMV expression (70 ± 4.2%, 65 ± 3.4%, and 82 ± 3.6% inhibition of viral expression, respectively, n = 5). In contrast, no viral suppression was observed following IL-6 treatment. Suppressive activity was dependent on the addition of cytokines before CMV infection. Cytokine pretreatment did not affect CMV entry into primary astrocytes, and the observed cytokine-induced suppressive activity was not affected by the NO synthase inhibitor NG-monomethyl-l-arginine (NGMA). Instead, the suppressive effect appeared to be mediated through a mechanism involving inhibition of CMV major immediate early promoter activity. These results support the hypothesis that proinflammatory cytokines possess anti-CMV activity in brain cells and may lead to new interventions for CMV encephalitis based upon immunotherapy.
Identification of Multiple Simian Immunodeficiency Virus (SIV)-Specific CTL Epitopes in Sooty Mangabeys with Natural and Experimentally Acquired SIV Infection
Authors: Kaur Amitinder , Yang Janet , Hempel Donna , Gritz Linda , Mazzara Gail P. , McClure Harold , Johnson R. Paul
Published: April 2014

Host immune responses to SIV infection in sooty mangabeys are likely to be an important determinant of how such nonhuman primate species maintain asymptomatic lentivirus infection. We have previously described two patterns of asymptomatic SIV infection in sooty mangabeys: low viral loads with vigorous SIV-specific CTL activity in SIVmac239-infected sooty mangabeys, and high viral loads with generally weak or absent SIV-specific CTL activity in naturally infected sooty mangabeys. To define the specificity of the CTL response in SIV-infected mangabeys, we characterized CTL epitopes in two naturally infected and three SIVmac239-infected sooty mangabeys. Compared with that in SIVmac239-infected mangabeys, the yield of SIV-specific CTL clones was significantly lower in naturally infected sooty mangabeys. All CTL clones were phenotypically CD3+ CD8+, and lysis was MHC restricted. Seven SIV CTL epitopes were identified in five sooty mangabeys: one in Gag and three each in Nef and Envelope (Env). The CTL epitopes mapped to conserved regions in the SIV genome and were immunodominant. Several similar or identical CTL epitopes were recognized by both naturally infected and SIVmac239-infected mangabeys that shared class I MHC alleles. To our knowledge, this is the first report of SIV-specific CTL epitopes in sooty mangabeys. Longitudinal studies of viral load and sequence variation in CTL epitopes may provide useful information on the role of CTL in control or persistence of SIV infection in sooty mangabeys.
Mechanism and Function of a Newly Identified CpG DNA Motif in Human Primary B Cells
Authors: Hartmann Gunther , Krieg Arthur M.
Published: April 2014

The vertebrate immune system recognizes bacterial DNA based on the presence of unmethylated CpG-dinucleotides in particular base contexts (“CpG motifs”). In contrast to mice, knowledge about CpG-mediated effects on human B cells is poor. In the present study we identify and determine an optimal human CpG motif. A phosphodiester oligonucleotide containing this motif strongly stimulated CD86, CD40, CD54, and MHC class II expression, IL-6 synthesis, and proliferation of primary human B cells. These effects required internalization of the oligonucleotide and endosomal maturation. The molecular mechanism of action of this CpG motif was associated with the sustained induction of the NF-κB p50/p65 heterodimer and of the transcription-factor complex AP-1. Transcription-factor activation by CpG DNA was preceded by increased phosphorylation of the stress kinases c-Jun N-terminal kinase and p38, and of activating transcription factor-2. In contrast to CpG, signaling through the B cell receptor led to activation of extracellular receptor kinase and to phosphorylation of a different isoform of c-Jun N-terminal kinase. These studies define the structure of a highly active human CpG motif and characterize its molecular mechanism of action in primary human B cells.
Ebola Virus Secretory Glycoprotein (sGP) Diminishes FcγRIIIB-to-CR3 Proximity on Neutrophils
Authors: Kindzelskii Andrei L. , Yang Zhi-yong , Nabel Gary J. , Todd Robert F. , Petty Howard R.
Published: April 2014

Previous studies have shown that Ebola virus’ secretory glycoprotein (sGP) binds to FcγRIIIB (CD16b) and inhibits L-selectin shedding. In this study, we test the hypothesis that sGP interferes with the physical linkage between CR3 and FcγRIIIB. Neutrophils were stained with rhodamine-conjugated anti-CD16b mAb (which does not inhibit sGP binding) and fluorescein-conjugated anti-CR3 mAb reagents and then incubated in media with or without sGP. Physical proximity between fluorochrome-labeled CR3 and FcγRIIIB on individual cells was measured by resonance energy transfer (RET) imaging, quantitative RET microfluorometry, and single-cell imaging spectrophotometry. Cells incubated with control supernatants displayed a significant RET signal, indicative of physical proximity (<7 nm) between CR3 and FcγRIIIB. In contrast, cells exposed to sGP showed a significant reduction in the CR3-FcγRIIIB RET signal using these methods. Interestingly, colocalization and cocapping of CR3 and FcγRIIIB were not affected, suggesting that the proximity of these two receptors is reduced without triggering dissociation. Thus, sGP alters the physical linkage between FcγRIIIB and CR3.
Activation of Human Neutrophils byMycobacterium tuberculosisH37Ra Involves Phospholipase Cγ2, Shc Adapter Protein, and p38 Mitogen-Activated Protein Kinase
Authors: Perskvist Nasrin , Zheng Limin , Stendahl Olle
Published: April 2014

Recent studies have shown that human neutrophils play a significant protective role in mycobacteria infection. When encountered with mycobacteria, neutrophils exhibit the typical early bactericidal responses including phagocytosis and generation of reactive oxygen intermediates (ROI), but the underlying mechanisms are largely unknown. The present study shows that stimulation of neutrophils with an attenuated strain of Mycobacterium tuberculosis H37Ra (Mtb) led to a tyrosine kinase-dependent ROI production in these cells. Stimulation with Mtb induces a rapid and transient tyrosine phosphorylation of several proteins, one of which was identified as phospholipase Cγ2 (PLCγ2). Several tyrosine-phosphorylated proteins were associated with the PLCγ2 precipitates from Mtb-stimulated neutrophils, of which pp46 was characterized as the Shc adapter protein. A role for PLCγ2-Shc association in the generation of ROI is supported by the observations that stimulation with Mtb causes the activation of p38 mitogen-activated protein kinase (MAPK), a downstream target of the Shc/Ras signaling cascade, and that the effect of genistein on ROI production coincided with its ability to inhibit both PLCγ2-Shc association and p38 MAPK activation. Moreover, pretreatment of neutrophils with a PLC inhibitor markedly suppresses the Mtb-stimulated ROI production as well as p38 MAPK activation in these cells. Taken together, these results indicate that stimulation of neutrophils with Mtb triggers the tyrosine phosphorylation of PLCγ2 and its association with Shc, and that such association is critical for the Mtb-stimulated ROI production through activating p38 MAPK.
Lipopolysaccharide Activates Distinct Signaling Pathways in Intestinal Epithelial Cell Lines Expressing Toll-Like Receptors
Authors: Cario Elke , Rosenberg Ian M. , Brandwein Steven L. , Beck Paul L. , Reinecker Hans-Christian , Podolsky Daniel K.
Published: April 2014

LPS elicits several immediate proinflammatoy responses in peripheral blood leukocytes via a recently described pathway including CD14, Toll-like receptors (TLR), serine-threonine kinases, and NF-κB transcription factor. However, the functional responses of intestinal epithelial cells (IEC) to stimulation with LPS are unknown. Expression of mRNA and protein for CD14 and TLRs were assessed by RT-PCR, immunoblotting, and immunohistochemistry in mouse and human IEC lines. LPS-induced activation of signaling pathways (p42/p44 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK), p38, p65, NF-κB) were assessed by immunoblotting and gel shifts. CD14 mRNA and protein expression were not detectable in IEC. However, human TLR2, TLR3, and TLR4 mRNA were present in IEC. TLR4 protein was expressed in all cell lines; however, TLR2 protein was absent in HT29 cells. Immunofluorescent staining of T84 cells demonstrated the cell-surface presence of the TLRs. LPS-stimulation of IEC resulted in activation (>1.5-fold) of the three members of the MAPK family. In contrast, LPS did not significantly induce activation of JNK and p38 in CMT93 cells, p38 in T84 cells and MAPK and JNK in HT29 cells. Downstream, LPS activated NF-κB in IEC in a time-, dose-, and serum-dependent manner. IEC express TLRs that appear to mediate LPS stimulation of specific intracellular signal transduction pathways in IEC. Thus, IEC may play a frontline role in monitoring lumenal bacteria.
CpG Oligonucleotides Can Prophylactically Immunize Against Th2-Mediated Schistosome Egg-Induced Pathology by an IL-12-Independent Mechanism
Authors: Chiaramonte Mónica G. , Hesse Matthias , Cheever Allen W. , Wynn Thomas A.
Published: April 2014

Using a Schistosoma mansoni egg-induced granuloma model, we examined the ability of CpG oligodeoxynucleotides (ODN) to suppress Th2-type cytokine expression and to prophylactically immunize against Th2-dependent pulmonary pathology. The mechanism was examined by studying Th2 response regulation in cytokine-deficient mice. Surprisingly, our findings revealed several functions of CpG DNA that were completely IL-12 independent. Most striking was the marked suppression in Th2 cytokine expression and granulomatous inflammation observed in egg/CpG-sensitized IL-12-deficient mice. Immune deviation was not dependent on NK or B cells. However, a role for IL-10, B7.1, and CD40 expression in Th2 response inhibition was suggested. Indeed, CpG ODN up-regulated all three elements in both wild-type and IL-12-deficient mice. The role of IL-10 was demonstrated in mice exhibiting combined deficiencies in IL-12 and IL-10. Here, a marked increase in egg-specific IL-4/IL-5-producing cells confirmed a role for both cytokines in Th2 response inhibition. Nevertheless, the frequency of Th2-producing cells was again reduced by CpG ODN. However, in marked contrast to IL-12-deficient animals, a significant increase in IFN-γ-producing cells likely explains the reduced Th2 response in IL-10/IL-12-deficient mice. Thus, a novel IL-12-independent type 1-inducing pathway was revealed in the combined absence of IL-12 and IL-10. Together, these data demonstrate 1) that the Th1-promoting activity of CpG DNA is controlled by IL-12 and IL-10, and 2) that Th2 response inhibition by CpG ODN involves IL-12-independent changes in IL-10 and costimulatory molecule expression. These findings illustrate the utility of CpG DNA as adjuvants for vaccines designed to prevent Th2-dependent immunopathology.
Characterization of CD4+ T Cell Responses During Natural Infection with Salmonella typhimurium
Authors: McSorley Stephen J. , Cookson Brad T. , Jenkins Marc K.
Published: April 2014

CD4+ T cells are important for resistance to infection with Salmonella typhimurium. However, the Ag specificity of this T cell response is unknown. Here, we demonstrate that a significant fraction of Salmonella-specific CD4+ T cells respond to the flagellar filament protein, FliC, and that this Ag has the capacity to protect naive mice from lethal Salmonella infection. To characterize this Ag-specific response further, we generated FliC-specific CD4+ T cell clones from mice that had resolved infection with an attenuated strain of Salmonella. These clones were found to respond to an epitope from a constant region of FliC, enabling them to cross-react with flagellar proteins expressed by a number of distinct Salmonella serovars.
Compartmental Differences in NK Cell Responsiveness to IL-12 During Lymphocytic Choriomeningitis Virus Infection
Authors: Pien Gary C. , Biron Christine A.
Published: April 2014

Some, but not all, viral infections induce endogenous IL-12 to drive NK cell IFN-γ production and downstream antiviral defenses during innate immune responses. Even though lymphocytic choriomeningitis virus (LCMV) can be sensitive to IFN-γ-mediated antiviral effects, infections with this agent do not elicit IL-12 or early IFN-γ in immunocompetent hosts. Studies presented here demonstrate that LCMV infections of mice not only fail to induce IL-12, but also modify responsiveness to exogenous IL-12 for IFN-γ production. IFN-γ responses induced by IL-12 administration were greatly diminished in splenic populations, but significantly increased in serum and hepatic leukocytes, during the early course of LCMV infections. The IFN-γ production was NK cell dependent, and the compartmental dichotomy between spleen and liver was also demonstrated in response to in vitro IL-12 stimulation. Although infections did increase proportions and numbers of liver NK cells, changes in responsiveness for IFN-γ expression could not be explained by cell redistribution. Corroborating changes in proportions of NK cells induced to express intracellular IFN-γ protein within the compartments were observed. The reduction in ability of splenic populations to produce IL-12-induced IFN-γ after infection by LCMV was associated with decreased efficacy of administered IL-12 for promoting IFN-γ-dependent antiviral effects in the spleen. Concomitantly, the maintenance of hepatic population IFN-γ production was associated with preserved efficacy of administered IL-12 to elicit IFN-γ-dependent antiviral effects in the liver. Taken together, these results demonstrate modifications of compartmental responses to IL-12 by viral infections and the consequences of these changes for efficacy of cytokine therapy.
Disruption of T Cell Tolerance to Self-Immunoglobulin Causes Polyclonal B Cell Stimulation Followed by Inactivation of Responding Autoreactive T Cells
Authors: Choudhury Arpita , Mukherjee Paushali , Basu Sandip K. , George Anna , Rath Satyajit , Bal Vineeta
Published: April 2014

Scavenger receptor (SR)-specific delivery by maleylation of a ubiquitous self-protein, Ig, to SR-bearing APCs results in self-limiting induction of autoimmune effects in vivo. Immunization with maleyl-Ig breaks T cell tolerance to self-Ig and causes hypergammaglobulinemia, with increases in spleen weight and cellularity. The majority of splenic B cells show an activated phenotype upon maleyl-Ig immunization, leading to large-scale conversion to a CD138+ phenotype and to significant increases in CD138-expressing splenic plasma cells. The polyclonal B cell activation, hypergammaglobulinemia, and autoreactive Ig-specific T cell responses decline over a 2-mo period postimmunization. Following adoptive transfer, T cells from maleyl-Ig-immune mice taken at 2 wk postimmunization can induce hypergammaglobulinemia in the recipients, but those taken at 10 wk postimmunization cannot. Hypergammaglobulinemia in the adoptive transfer recipients is also transient and is followed by an inability to respond to fresh maleyl-Ig immunization, suggesting that the autoreactive Ig-specific T cells are inactivated peripherally following disruption of tolerance. Thus, although autoreactive T cell responses to a ubiquitous self-Ag, Ig, are induced by SR-mediated delivery to professional APCs in vivo resulting in autoimmune pathophysiological effects, they are effectively and rapidly turned off by inactivation of these activated Ig-specific T cells in vivo.
Inhibition of IL-12 Production in Human Monocyte-Derived Macrophages by TNF
Authors: Ma Xiaojing , Sun Junwei , Papasavvas Emmanouil , Riemann Helge , Robertson Susan , Marshall Jason , Bailer Robert T. , Moore Anne , Donnelly Raymond P. , Trinchieri Giorgio , Montaner Luis J.
Published: April 2014

IL-12 is a pivotal cytokine that links the innate and adaptive immune responses. TNF-α also plays a key role in orchestrating inflammation and immunity. The reciprocal influence of these two inflammatory mediators on each other may have significant impact on the cytokine balance that shapes the type and extent of immune responses. To investigate the relationship between TNF-α and IL-12 production, we analyzed the effects of exposure of human monocyte-derived macrophages to TNF-α on LPS- or Staphylococcus aureus-induced IL-12 production in the presence or absence of IFN-γ. TNF-α is a potent inhibitor of IL-12 p40 and p70 secretion from human macrophages induced by LPS or S. aureus. IL-10 is not responsible for the TNF-α-mediated inhibition of IL-12. TNF-α selectively inhibits IL-12 p40 steady-state mRNA, but not those of IL-12 p35, IL-1α, IL-1β, or IL-6. Nuclear run-on analysis identified this specific inhibitory effect at the transcriptional level for IL-12 p40 without down-regulation of the IL-12 p35 gene. The major transcriptional factors identified to be involved in the regulation of IL-12 p40 gene expression by LPS and IFN-γ, i.e., c-Rel, NF-κB p50 and p65, IFN regulatory factor-1, and ets-2, were not affected by TNF-α when examined by nuclear translocation and DNA binding. These data demonstrate a selective negative regulation on IL-12 by TNF-α, identifying a direct negative feedback mechanism for inflammation-induced suppression of IL-12 gene expression.
TCR-Independent Activation of Extrathymically Developed, Self Antigen-Specific T Cells by IL-2/IL-15
Authors: Yamada Hisakata , Nakamura Takahiko , Matsuzaki Goro , Iwamoto Yukihide , Nomoto Kikuo
Published: April 2014

Naive intrathymically developed T cells, which express foreign Ag-specific TCR, do not express IL-2R. After antigenic stimulation, they express high affinity IL-2R, which enables IL-2 to be used as an autocrine growth factor. On the contrary, extrathymically developed T cells, which express self Ag-specific TCR but are unresponsive to antigenic stimulation, spontaneously express low affinity IL-2R. In this study, we compared the responses of these two subsets of T cells to IL-2R stimulation and examined the influences of TCR-mediated signaling on the responses. IL-2 or IL-15 augmented the proliferative response of Ag-stimulated, intrathymically developed T cells. On the other hand, extrathymically developed T cells proliferated in response to IL-2 or IL-15, independently of Ag stimulation. Furthermore, both IL-2 and IL-15 induced IFN-γ production of these T cells, which is strikingly augmented by the presence of IL-12. These results revealed functional differences between intrathymically developed, foreign Ag-specific T cells and extrathymically developed, self Ag-specific T cells. The latter can be activated by some inflammatory cytokines, in an Ag-independent manner, similar to NK cells.
Signals from the IL-9 Receptor Are Critical for the Early Stages of Human Intrathymic T Cell Development
Authors: De Smedt Magda , Verhasselt Bruno , Kerre Tessa , Vanhecke Dominique , Naessens Evelien , Leclercq Georges , Renauld Jean-Christophe , Van Snick Jacques , Plum Jean
Published: April 2014

Highly purified human CD34+ hemopoietic precursor cells differentiate into mature T cells when seeded in vitro in isolated fetal thymic lobes of SCID mice followed by fetal thymus organ culture (FTOC). Here, this chimeric human-mouse FTOC was used to address the role of IL-9 and of the α-chain of the IL-9 receptor (IL-9Rα) in early human T cell development. We report that addition of the mAb AH9R7, which recognizes and blocks selectively the human high affinity α-chain of the IL-9R, results in a profound reduction of the number of human thymocytes. Analysis of lymphoid subpopulations indicates that a highly reduced number of cells undergo maturation from CD34+ precursor cells toward CD4+CD3−CD8−CD1+ progenitor cells and subsequently toward CD4+CD8+ double positive (DP) thymocytes. Addition of IL-9 to the FTOC resulted in an increase in cell number, without disturbing the frequencies of the different subsets. These data suggest that IL-9Rα signaling is critical in early T lymphoid development.
Ly49I NK Cell Receptor Transgene Inhibition of Rejection of H2b Mouse Bone Marrow Transplants
Authors: Liu Jingxuan , Morris Margaret A. , Nguyen Paul , George Thaddeus C. , Koulich Elena , Lai Wayne C. , Schatzle John D. , Kumar Vinay , Bennett Michael
Published: April 2014

The Ly49 family of genes encode NK cell receptors that bind class I MHC Ags and transmit negative signals if the cytoplasmic domains have immunoregulatory tyrosine-based inhibitory motifs (ITIMs). 5E6 mAbs recognize Ly49C and Ly49I receptors and depletion of 5E6+ NK cells prevents rejection of allogeneic or parental-strain H2d bone marrow cell (BMC) grafts. To determine the function of the Ly49I gene in the rejection of BMC grafts, we transfected fertilized eggs of FVB mice with a vector containing DNA for B6 strain Ly49I (Ly49IB6). Ly49IB6 is ITIM+ and is recognized by 5E6 as well as Ly49I-specific 8H7 mAbs. Normal FVB H2q mice reject H2b but not H2d BMC allografts, and the rejection of H2b BMC was inhibited partially by anti-NK1.1 and completely by anti-asialo GM1, but not by anti-CD8, Abs. In FVB mice, NK1.1 is expressed on only 60% NK cells. FVB.Ly49IB6 hosts failed to reject H2d or H2b BMC, but did reject class I-deficient TAP-1−/− BMC, indicating that NK cells were functional. Nondepleting doses of anti-Ly49I Abs reversed the acceptance of H2b BMC by FVB.Ly49IB6 mice. FVB.Ly49IB6+/− mice were crossed and back-crossed with 129 mice—H2b, 5E6−, poor responders to H2d BMC grafts. While transgene-negative H2b/q F1 or first-generation back-crossed mice rejected H2b marrow grafts (hybrid resistance), transgene-positive mice did not. Thus B6 strain Ly49I receptors transmit inhibitory signals from H2b MHC class I molecules. Moreover, Ly49IB6 has no positive influence on the rejection of H2d allografts.
The Balance of Protein Kinase C and Calcium Signaling Directs T Cell Subset Development
Authors: Noble Alistair , Truman Jean Philip , Vyas Beejal , Vukmanovic-Stejic Milica , Hirst William J. , Michael Kemeny David
Published: April 2014

Development of naive T cells into type 1 (Th1, Tc1) or type 2 (Th2, Tc2) effector cells is thought to be under the control of cytokines. In this study, we show that when both IL-12 and IL-4 are present, murine and human T cell differentiation is regulated by the balance of protein kinase C (PKC) and calcium signaling within T cells. Although both biochemical signals were required for T cell activation via the TCR, altering the balance between them redirected type 1 cells to type 2 and vice versa. Stimulation of calcium signaling or inhibition of PKC favored type 1 differentiation, whereas stimulation of PKC or inhibition of calcineurin resulted in type 2 effectors. Altered peptide ligands induced distinct balances of PKC/calcium signaling and altered Tc1/Tc2 development in TCR-transgenic CD8 T cells. The data suggest novel strategies for manipulation of the immune response in vivo.
Abrogation of CTL Epitope Processing by Single Amino Acid Substitution Flanking the C-Terminal Proteasome Cleavage Site
Authors: Beekman Nico J. , van Veelen Peter A. , van Hall Thorbald , Neisig Anne , Sijts Alice , Camps Marcel , Kloetzel Peter-M. , Neefjes Jacques J. , Melief Cornelis J. , Ossendorp Ferry
Published: April 2014

CTL directed against the Moloney murine leukemia virus (MuLV) epitope SSWDFITV recognize Moloney MuLV-induced tumor cells, but do not recognize cells transformed by the closely related Friend MuLV. The potential Friend MuLV epitope has strong sequence homology with Moloney MuLV and only differs in one amino acid within the CTL epitope and one amino acid just outside the epitope. We now show that failure to recognize Friend MuLV-transformed tumor cells is based on a defect in proteasome-mediated processing of the Friend epitope which is due to a single amino acid substitution (N→D) immediately flanking the C-terminal anchor residue of the epitope. Proteasome-mediated digestion analysis of a synthetic 26-mer peptide derived from the Friend sequence shows that cleavage takes place predominantly C-terminal of D, instead of V as is the case for the Moloney MuLV sequence. Therefore, the C terminus of the epitope is not properly generated. Epitope-containing peptide fragments extended with an additional C-terminal D are not efficiently translocated by TAP and do not show significant binding affinity to MHC class I-Kb molecules. Thus, a potential CTL epitope present in the Friend virus sequence is not properly processed and presented because of a natural flanking aspartic acid that obliterates the correct C-terminal cleavage site. This constitutes a novel way to subvert proteasome-mediated generation of proper antigenic peptide fragments.
The Tumor Suppressor PTEN Regulates T Cell Survival and Antigen Receptor Signaling by Acting as a Phosphatidylinositol 3-Phosphatase
Authors: Wang Xiaodong , Gjörloff-Wingren Anette , Saxena Manju , Pathan Nuzhat , Reed John C. , Mustelin Tomas
Published: April 2014

The tumor suppressor gene PTEN encodes a 55-kDa enzyme that hydrolyzes both protein phosphotyrosyl and 3-phosphorylated inositol phospholipids in vitro. We have found that the latter activity is physiologically relevant in intact T cells. Expression of active PTEN lead to a 50% loss of transfected cells due to increased apoptosis, which was completely prevented by coexpression of a constitutively active, membrane-bound form of protein kinase B. A mutant of PTEN selectively lacking lipid phosphatase activity, but retaining protein phosphatase activity, had no effects on cell number. Active (but not mutant) PTEN also decreased TCR-induced activation of the mitogen-activated protein kinase ERK2 (extracellular signal-related kinase 2), as seen after inhibition of phosphatidylinositol 3-kinase. Our data indicate that PTEN is a phosphatidylinositol 3-phosphatase in T cells, and we suggest that PTEN may play a role in the regulation of T cell survival and TCR signaling by directly opposing phosphatidylinositol 3-kinase.
A Prominent Role for Sp1 During Lipopolysaccharide- Mediated Induction of the IL-10 Promoter in Macrophages
Authors: Brightbill Hans D. , Plevy Scott E. , Modlin Robert L. , Smale Stephen T.
Published: April 2014

IL-10 is an antiinflammatory cytokine secreted by activated macrophages and Th2 cells. IL-10 secretion promotes the down-regulation of proinflammatory cytokine synthesis and the development of Th2 responses. In macrophages, proinflammatory cytokines appear to be induced by similar mechanisms, but the IL-10 induction mechanisms have not been examined. We have analyzed the murine IL-10 promoter in the RAW264.7 macrophage line activated with LPS. A comprehensive mutant analysis revealed only one element upstream of the core promoter that was essential for promoter induction. A refined mutant analysis localized this element to nucleotides −89 to −78, and gel shift experiments revealed that it represents a nonconsensus binding site for Sp1. The functional relevance of Sp1 was supported by the high affinity of the interaction, the close correlation between the nucleotides required for Sp1 binding and promoter function, and the ability of an Sp1 consensus sequence to substitute for the −89/−78 promoter sequence. Evidence that Sp1 may be a target of signaling pathways involved in IL-10 induction was provided by the exclusive requirement for the Sp1 binding site, by the ability of the Sp1 site to confer induction to a heterologous promoter, and by the delineation of an Sp1 domain that can mediate induction. No relevant contribution from Rel, C/EBP (CCAAT/enhancer-binding protein), or AP-1 binding sites, which regulate most proinflammatory cytokine promoters, was observed. Together, these results demonstrate that IL-10 gene regulation is distinct from the regulation of proinflammatory cytokine genes, and suggest that Sp1 may be a central mediator of IL-10 induction.
Role of Tumor Cell Apoptosis in Tumor Antigen Migration to the Draining Lymph Nodes
Authors: Bonnotte Bernard , Favre Nathalie , Moutet Monique , Fromentin Annie , Solary Eric , Martin Monique , Martin François
Published: April 2014

Establishment of an immune response against cancer may depend on the capacity of dendritic cells to transfer tumor Ags into T cell-rich areas. To check this possibility, we used a colon cancer cell variant that yields tumors undergoing complete T cell-dependent rejection when injected into syngeneic rats. We previously demonstrated that immunogenicity of these tumors depended on the early apoptosis of a part of these tumor cells. In this paper we show that fluorescent tumor cell proteins are released from FITC-labeled tumor cells and undergo engulfment by tumor-infiltrating monocytes without a phenotype of mature dendritic cells or macrophages. Fluorescence-labeled mononuclear cells with a phenotype of MHC class II+ dendritic cells are also found in the T cell areas of the draining lymph nodes. Interestingly, no fluorescent cell can be found in lymph nodes after a s.c. injection of Bcl2-transfected apoptosis-resistant tumor cells that yielded progressive tumors. Proliferation of tumor-immune T lymphocytes was induced by dendritic cells isolated from the draining lymph nodes recovered after a s.c. injection of apoptosis-sensitive, but not apoptosis-resistant, tumor cells. These results show that tumor cell apoptosis releases proteins that are engulfed by inflammatory cells in the tumor, then transported to lymph node T cell areas where they can induce a specific immune response leading to tumor rejection.