bluuebunny/crossref_metadata_2025 huggingface dataset:
The Linker Phosphorylation Site Tyr292 Mediates the Negative Regulatory Effect of Cbl on ZAP-70 in T Cells
Authors: Rao Navin , Lupher Mark L. , Ota Satoshi , Reedquist Kris A. , Druker Brian J. , Band Hamid
Published: April 2014

The protooncogene product Cbl has emerged as a negative regulator of tyrosine kinases. We have shown previously that Cbl binds to ZAP-70 through its N-terminal tyrosine kinase binding (TKB) domain. In this study, we demonstrate that overexpression of Cbl in Jurkat T cells decreases the TCR-induced phosphorylation of ZAP-70 and other cellular phosphoproteins. Coexpression of Cbl with ZAP-70 in COS cells reproduced the Cbl-induced reduction in the level of phosphorylated ZAP-70. The effect of Cbl was eliminated by the TKB-inactivating G306E mutation in Cbl as well as by a phenylalanine mutation of Tyr292 within the TKB domain binding site on ZAP-70. Notably, the oncogenic Cbl-70Z/3 mutant associated with ZAP-70, but did not reduce the levels of phosphorylated ZAP-70. Overexpression of Cbl, but not Cbl-G306E, in Jurkat T cells led to a decrease in the TCR-induced NF-AT luciferase reporter activity. Overexpression of the TKB domain itself, but not its G306E mutant, functioned in a dominant-negative manner and led to an increase in NF-AT reporter activity. Cbl-70Z/3-overexpressing cells exhibited an increase in both basal and TCR-induced NF-AT luciferase reporter activity, and this trend was reversed by the G306E mutation. Finally, by reconstituting a ZAP-70-deficient Jurkat T cell line, p116, we demonstrate that wild-type ZAP-70 is susceptible to the negative regulatory effect of Cbl, whereas the ZAP-70-Y292F mutant is resistant. Together, our results establish that the linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells.
Cytoprotection of Human Umbilical Vein Endothelial Cells Against Apoptosis and CTL-Mediated Lysis Provided by Caspase-Resistant Bcl-2 Without Alterations in Growth or Activation Responses
Authors: Zheng Lian , Dengler Thomas J. , Kluger Martin S. , Madge Lisa A. , Schechner Jeffrey S. , Maher Stephen E. , Pober Jordan S. , Bothwell Alfred L. M.
Published: April 2014

Graft endothelial cells are primary targets of host CTL-mediated injury in acute allograft rejection. As an in vitro trial of gene therapy to reduce CTL-mediated endothelial injury, we stably transduced early passage HUVEC with a caspase-resistant mutant form (D34A) of the anti-apoptotic gene Bcl-2. Bcl-2 transductants were compared with HUVEC transduced in parallel with an enhanced green fluorescent protein (EGFP) gene. Both transduced HUVEC have equivalent growth rates in complete medium and both show contact inhibition of growth. However, compared with EGFP-transduced HUVEC, the Bcl-2-transduced cells are resistant to the apoptotic effects of serum and growth factor withdrawal and are also resistant to the induction of apoptosis by staurosporine or by ceramide, with or without TNF. Transduced Bcl-2 did not reduce TNF-mediated NF-κB activation or constitutive expression of class I MHC molecules. HUVEC expressing D34A Bcl-2 were significantly more resistant to lysis by either class I-restricted alloreactive or PHA-redirected CTL than were HUVEC expressing EGFP. We conclude that transduction of graft endothelial cells with D34A Bcl-2 is a possible approach for reducing allograft rejection.
Cyclic Nucleotide Phosphodiesterase 3B Is a Downstream Target of Protein Kinase B and May Be Involved in Regulation of Effects of Protein Kinase B on Thymidine Incorporation in FDCP2 Cells
Authors: Ahmad Faiyaz , Cong Li-Na , Stenson Holst Lena , Wang Ling-Mei , Rahn Landstrom Tova , Pierce Jaclyn H. , Quon Michael J. , Degerman Eva , Manganiello Vincent C.
Published: April 2014

Wild-type (F/B), constitutively active (F/B*), and three kinase-inactive (F/Ba−, F/Bb−, F/Bc−) forms of Akt/protein kinase B (PKB) were permanently overexpressed in FDCP2 cells. In the absence of insulin-like growth factor-1 (IGF-1), activities of PKB, cyclic nucleotide phosphodiesterase 3B (PDE3B), and PDE4 were similar in nontransfected FDCP2 cells, mock-transfected (F/V) cells, and F/B and F/B− cells. In F/V cells, IGF-1 increased PKB, PDE3B, and PDE4 activities ∼2-fold. In F/B cells, IGF-1, in a wortmannin-sensitive manner, increased PKB activity ∼10-fold and PDE3B phosphorylation and activity (∼4-fold), but increased PDE4 to the same extent as in F/V cells. In F/B* cells, in the absence of IGF-1, PKB activity was markedly increased (∼10-fold) and PDE3B was phosphorylated and activated (3- to 4-fold); wortmannin inhibited these effects. In F/B* cells, IGF-1 had little further effect on PKB and activation/phosphorylation of PDE3B. In F/B− cells, IGF-1 activated PDE4, not PDE3B, suggesting that kinase-inactive PKB behaved as a dominant negative with respect to PDE3B activation. Thymidine incorporation was greater in F/B* cells than in F/V cells and was inhibited to a greater extent by PDE3 inhibitors than by rolipram, a PDE4 inhibitor. In F/B cells, IGF-1-induced phosphorylation of the apoptotic protein BAD was inhibited by the PDE3 inhibitor cilostamide. Activated PKB phosphorylated and activated rPDE3B in vitro. These results suggest that PDE3B, not PDE4, is a target of PKB and that activated PDE3B may regulate cAMP pools that modulate effects of PKB on thymidine incorporation and BAD phosphorylation in FDCP2 cells.
A Switch in Distinct IκBα Degradation Mechanisms Mediates Constitutive NF-κB Activation in Mature B Cells
Authors: Fields Erika R. , Seufzer Bradley J. , Oltz Eugene M. , Miyamoto Shigeki
Published: April 2014

Inducible activation of cytoplasmic NF-κB/Rel transcription factors occurs via proteasome-dependent degradation of an associated inhibitor, termed IκBα. Mature B lymphocytes constitutively express nuclear NF-κB, which is important for their long-term survival. The intrinsic mechanisms by which B cells constitutively activate NF-κB are unknown. In this paper we demonstrate that maintenance of NF-κB activity in primary B cells is mediated by a novel calcium-dependent, but proteasome-independent, mechanism. Moreover, we show that differentiation of conditionally transformed pre-B cells is accompanied by a switch from proteasome-dependent to proteasome-independent degradation of IκBα. Our findings indicate that IκBα degradation mechanisms are dynamic during B cell development, and ultimately establish constitutive NF-κB activity in mature B lymphocytes.
Synthetic Endotoxin-Binding Peptides Block Endotoxin- Triggered TNF-α Production by Macrophages In Vitro and In Vivo and Prevent Endotoxin-Mediated Toxic Shock
Authors: Dankesreiter Silke , Hoess Adolf , Schneider-Mergener Jens , Wagner Hermann , Miethke Thomas
Published: April 2014

Lipid A, the conserved portion of endotoxin, is the major mediator of septic shock; therefore, endotoxin-neutralizing molecules could have important clinical applications. Here we show that peptides derived from Limulus anti-LPS factor (LALF), bactericidal/permeability increasing protein (BPI) and endotoxin-binding protein, bind to lipid A and block the recombinant LALF/lipid A interaction in vitro. Because their neutralizing capacity in vitro as well as in vivo has been limited, we created hybrid peptides comprising two endotoxin-binding domains. The hybrid molecule LL-10-H-14, containing endotoxin-binding domains from LALF and endotoxin-binding protein, turned out to be the most active peptide within the series of peptides tested here to inhibit the CD14/lipid A interaction and is able in vitro to block the endotoxin-induced TNF-α release of murine macrophages up to 90%. Furthermore, LL-10-H-14 not only reduced peak serum levels of TNF-α of mice when preinjected but also reduced TNF-α levels when given 15 min after the endotoxin challenge. As compared with other peptides, only LL-10-H-14 is able to strongly decrease endotoxin-stimulated TNF-α release by human macrophage cell lines as well as by PBMC. Furthermore, the hybrid peptide is protective against endotoxin-provoked lethal shock. As such, LL-10-H-14 could have prophylactic and/or therapeutic properties in humans for the management of septic shock.
Up-Regulation of VCAM-1 and Differential Expansion of β Integrin-Expressing T Lymphocytes Are Associated with Immunity to PulmonaryMycobacterium tuberculosisInfection
Authors: Feng Carl G. , Britton Warwick J. , Palendira Umaimainthan , Groat Natalie L. , Briscoe Helen , Bean Andrew G. D.
Published: April 2014

Immune responses rely on an intricate system of adhesion molecules to coordinate the homing and retention of lymphocytes in both secondary lymphoid tissues and at sites of infection. To define the events associated with pulmonary immune responses, the expression of endothelial addressins and integrins on T cells was analyzed during Mycobacterium tuberculosis infection. In infected lung, expression of endothelial VCAM-1, but not mucosal addressin cell adhesion molecule-1, was up-regulated from 4 wk postinfection and persisted to at least 12 wk. Subsequent analysis of the corresponding integrins expressed on lung CD4+ and CD8+ T cells revealed an accumulation of β1high/β7−/low, and to a lesser extent β7high, integrin-expressing T cells during infection. Examination of integrin heterodimers showed that while α4 integrin was predominantly expressed on β1high/β7−/low cells, αE integrin was primarily associated with β7high. The majority of activated/memory T cells recruited during infection expressed high levels of β1 integrin and undetectable or low levels of β7 integrin. These T cells were capable of producing IFN-γ, a cytokine crucial for controlling M. tuberculosis infection. Rapid expansion of β1high, β7−, and β7high T cell populations in the lung upon secondary mycobacterial infection indicates the participation of these populations in the acquired immune response to the infection. Furthermore, treatment of infected mice with mAb to α4 or α4β7 integrin led to a reduction in lymphocytes and increase in granulocytes in the pulmonary infiltrate. These results reveal a crucial role for adhesion molecules in the generation of an effective pulmonary immune response to M. tuberculosis infection.
Apoptotic Thymocyte Clearance in Scavenger Receptor Class A-Deficient Mice Is Apparently Normal
Authors: Platt Nick , Suzuki Hiroshi , Kodama Tatsuhiko , Gordon Siamon
Published: April 2014

Studies of apoptotic cell uptake by phagocytes in vitro have implicated a number of different receptors capable of mediating ingestion. However, there is currently little evidence for involvement of any of these candidate receptors in vivo. Previously, we have shown by the use of a blocking mAb against the class A scavenger receptor (SR-A) and thymic macrophages prepared from SR-A null mice, that this receptor is responsible for ∼50% of the uptake of apoptotic thymocytes in vitro. In this study we have investigated the frequency of dying cells in the thymus of mice lacking SR-A. Our inability to demonstrate increased frequencies of nonphagocytosed Annexin V+, TUNEL+, or propidium iodide+ apoptotic thymocytes suggests there is no deficiency in apoptotic thymocyte clearance in these mice. Even when the rate of thymocyte apoptosis was increased by exposure of receptor-deficient mice to gamma irradiation, we did not detect a difference in the numbers of dying cells compared with similarly treated wild-type animals. This provides the first direct evidence of redundancy in apoptotic cell clearance mechanisms in vivo.
Long-Term Survival of Hamster Hearts in Presensitized Rats
Authors: Lin Yuan , Soares Miguel P. , Sato Koichiro , Csizmadia Eva , Robson Simon C. , Smith Neal , Bach Fritz H.
Published: April 2014

We transplanted hamster hearts into rats that had been sensitized to hamster cardiac grafts 5 days earlier as a model for discordant xenotransplantation. Sensitized rats had high serum levels of elicited anti-donor IgM and IgG that caused hyperacute rejection. Transient complement inhibition with cobra venom factor (CVF) plus daily and continuing cyclosporin A (CyA) prevented hyperacute rejection. However, grafts underwent delayed xenograft rejection (DXR). DXR involved IgG and associated Ab-dependent cell-mediated rejection, because depletion of IgG or Ab-dependent cell-mediated rejection-associated effector cells prolonged graft survival and the serum-mediated Ab-dependent cell-mediated cytotoxicity in vitro. Blood exchange in combination with CVF/CyA treatment dramatically decreased the level of preexisting Abs, but DXR still occurred in association with the return of Abs. Splenectomy and cyclophosphamide acted synergistically to delay Ab return, and when combined with blood exchange/CVF/CyA facilitated long-term survival of grafts. These grafts survived in the presence of anti-donor IgM, IgG, and complement that precipitated rejection of naive hearts, indicating that accommodation (survival in the presence of anti-graft Abs and complement) had occurred. We attribute the long-term survival to the removal of preexisting anti-donor Abs and therapy that attenuated the rate of Ab return. Under such conditions, the surviving hearts showed expression in endothelial cells and smooth muscle cells of protective genes and an intragraft Th2 immune response. Th2 responses and protective genes are associated with resistance to IgM- and IgG-mediated, complement-dependent and -independent forms of rejection.
Kinetics and Mechanism of ATP-Dependent IL-1β Release from Microglial Cells
Authors: Sanz Juana M. , Virgilio Francesco Di
Published: April 2014

Endotoxin-dependent release of IL-1β from mouse microglial cells is a very inefficient process, as it is slow and leads to accumulation of a modest amount of extracellular cytokine. Furthermore, secreted IL-1β is mostly in the procytokine unprocessed form. Addition of extracellular ATP to LPS-primed microglia caused a burst of release of a large amount of processed IL-1β. ATP had no effect on the accumulation of intracellular pro-IL-1β in the absence of LPS. In LPS-treated cells, ATP slightly increased the synthesis of pro-IL-1β. Optimal ATP concentration for IL-1β secretion was between 3 and 5 mM, but significant release could be observed at concentrations as low as 1 mM. At all ATP concentrations IL-1β release could be inhibited by increasing the extracellular K+ concentration. ATP-dependent IL-1β release was also inhibited by 90 and 60% by the caspase inhibitors YVAD and DEVD, respectively. Accordingly, in ATP-stimulated microglia, the p20 proteolytic fragment derived from activation of the IL-1-β-converting enzyme could be detected by immunoblot analysis. These experiments show that in mouse microglial cells extracellular ATP triggers fast maturation and release of intracellularly accumulated IL-β by activating the IL-1-β-converting enzyme/caspase 1.
Cutting Edge: Guinea Pigs with a Natural C3a-Receptor Defect Exhibit Decreased Bronchoconstriction in Allergic Airway Disease: Evidence for an Involvement of the C3a Anaphylatoxin in the Pathogenesis of Asthma
Authors: Bautsch Wilfried , Hoymann Heinz-Gerd , Zhang Qiuwang , Meier-Wiedenbach Ivo , Raschke Ursula , Ames Robert S. , Sohns Bettina , Flemme Nicole , Meyer zu Vilsendorf Andreas , Grove Melanie , Klos Andreas , Köhl Jörg
Published: April 2014

Asthma is a major cause of morbidity worldwide with prevalence and severity still increasing at an alarming pace. Hallmarks of this disease include early-phase bronchoconstriction with subsequent eosinophil infiltration, symptoms that may be mimicked in vivo by the complement-derived C3a anaphylatoxin, following its interaction with the single-copy C3aR. We analyzed the pathophysiological role of the C3a anaphylatoxin in a model of experimental OVA-induced allergic asthma, using an inbred guinea pig strain phenotypically unresponsive to C3a. Molecular analysis of this defect revealed a point mutation within the coding region of the C3aR that creates a stop codon, thereby effectively inactivating gene function. When challenged by OVA inhalation, sensitized animals of this strain exhibited a bronchoconstriction decreased by ∼30% in comparison to the corresponding wild-type strain. These data suggest an important role of C3a in the pathogenesis of asthma and define a novel target for drug intervention strategies.
CD73 Engagement Promotes Lymphocyte Binding to Endothelial Cells Via a Lymphocyte Function-Associated Antigen-1-Dependent Mechanism
Authors: Airas Laura , Niemelä Jussi , Jalkanen Sirpa
Published: April 2014

CD73 is a GPI-anchored lymphocyte adhesion molecule possessing an ecto-5′-nucleotidase enzyme activity. In this work, we show that engagement of lymphocyte CD73 increases lymphocyte binding to cultured endothelial cells (EC) in an LFA-1-dependent fashion. Engagement of CD73 by an anti-CD73 mAb 4G4 increases the adhesion of lymphocytes to cultured EC by about 80% compared with that of lymphocytes treated with a negative control Ab, and the increased adhesion can be blocked by an anti-CD18 mAb. The CD73-regulated increase in lymphocyte adhesion is not due to a conformational change leading to high-affinity LFA-1 receptors as assayed using mAb 24 against an activation-induced epitope of the molecule. Instead, CD73 engagement induces clustering of LFA-1 that is inhibitable by calpeptin, indicating involvement of Ca2+-dependent activation of a calpain-like enzyme in this process. In conclusion, the results shown here demonstrate that CD73 regulates the avidity of LFA-1 by clustering. This indicates a previously undescribed role for CD73 in controlling the poorly characterized activation step in the multistep cascade of lymphocyte extravasation. Moreover, these results suggest that in physiological conditions the activation step may result in clustering of LFA-1 rather than in an affinity change of the molecule.
Mac-1-Negative B-1b Phenotype of Natural Antibody-Producing Cells, Including Those Responding to Galα1,3Gal Epitopes in α1,3-Galactosyltransferase-Deficient Mice
Authors: Ohdan Hideki , Swenson Kirsten G. , Kruger Gray Huw S. , Yang Yong-Guang , Xu Yuanxin , Thall Aron D. , Sykes Megan
Published: April 2014

Human natural Abs against Galα1-3Galβ1-4GlcNAc (Gal) epitopes are a major barrier to xenotransplantation. Studies in this report, which use combined multiparameter flow cytometric sorting and enzyme-linked immunospot assay, demonstrate that anti-Gal IgM-producing cells are found exclusively in a small B cell subpopulation (i.e., CD21−/low IgMhigh B220low CD5− Mac-1− 493− cells) in the spleens of α1,3-galactosyltransferase-deficient mice. All IgM-producing cells were detected in a similar splenic subpopulation of α1,3-galactosyltransferase-deficient and wild-type mice. A higher frequency of B cells with anti-Gal surface IgM receptors was observed in the peritoneal cavity than in the spleen, but these did not actively secrete Abs, and showed phenotypic properties of B-1b cells (CD21−/low IgMhigh CD5− CD43+ Mac-1+). However, these became Mac-1− and developed anti-Gal Ab-producing activity after in vitro culture with LPS. The splenic B cells with anti-Gal receptors consisted of both Mac-1+ B-1b cells and Mac-1− B-1b-like cells. The latter comprised most anti-Gal IgM-producing cells. Our studies indicate that anti-Gal natural IgM Abs are produced by a B1b-like, Mac-1− splenic B cell population and not by plasma cells or B-1a cells. They are consistent with a model whereby B-1b cells lose Mac-1 expression upon Ag exposure and that these, rather than plasma cells, become the major IgM Ab-producing cell population.
Cyclic AMP Activates p38 Mitogen-Activated Protein Kinase in Th2 Cells: Phosphorylation of GATA-3 and Stimulation of Th2 Cytokine Gene Expression
Authors: Chen Chang-Hung , Zhang Dong-Hong , LaPorte Jody M. , Ray Anuradha
Published: April 2014

cAMP is an important second messenger with immunomodulatory properties. Elevation of intracellular cAMP in T cells, induced by agents such as IL-1α or PGs, inhibits T cell activation. In effector T cells, an increase in the level of intracellular cAMP inhibits cytokine production in Th1 cells but stimulates cytokine production in Th2 cells. Here we report that cAMP-induced effects in Th2 cells occur independently of the protein kinase A pathway, which is the major mediator of cAMP-induced signaling events in most cell types. Instead, cAMP stimulates activation of p38 mitogen-activated protein kinase in Th2 cells. This appears to be a Th2-selective event because cAMP barely increased p38 phosphorylation in Th1 cells. We show that in Th2 cells, cAMP promotes the production of both IL-5 and IL-13, which play distinct but critical roles in asthma pathogenesis. Our data also show that cAMP causes increased phosphorylation of the transcription factor GATA-3, which we have shown is a critical regulator of Th2 cytokine gene expression and, in turn, of airway inflammation in mice. Thus, Th2-specific GATA-3 expression and p38 mitogen-activated protein kinase activation together provide a molecular basis for the differential effects of cAMP in the two T helper cell subsets.
Characterization of the Heparin-Binding Properties of IL-6
Authors: Mummery Rosemary S. , Rider Christopher C.
Published: April 2014

We establish, using an ELISA approach, that recombinant human and murine IL-6 bind to an immobilized heparin-BSA complex. In the case of human IL-6, this binding is displaceable by soluble heparin, IC50 ∼2 μg/ml, corresponding to ∼200 nM. This binding is specific because chondroitin sulfates B and C fail to compete, whereas chondroitin sulfate A and several heparan sulfates are weak inhibitors. Of a range of chemically modified heparins examined, the strongest competitor was the 2-O-desulfated product, but even this showed a considerably reduced IC50 (∼30 μg/ml). The epitopes of five IL-6-specific mAbs were still accessible in heparin-bound IL-6, and the dimer formed from the association of rIL-6 with its truncated soluble receptor polypeptide, srIL-6α, still bound to heparin. Further analysis showed that heparin competed partially and weakly with the binding of srIL-6 to IL-6; however, it competed strongly for the binding of the rIL-6/srIL-6Rα dimer, to soluble glycoprotein 130. In studies of the proliferation of IL-6-sensitive Ba/F3 cells expressing glycoprotein 130, we were unable to detect any effect of either the removal of cell surface heparan sulfate, or addition of soluble heparin. By contrast, heparin was able to protect IL-6 from digestion by the bacterial endoproteinase Lys-C. Overall, our findings show that IL-6 is a heparin-binding cytokine. This interaction will tend to retain IL-6 close to its sites of secretion in the tissues by binding to heparin-like glycosaminoglycans, thus favoring a paracrine mode of activity. Moreover, this binding may serve to protect the IL-6 from proteolytic degradation.
Antagonism of Direct Alloreactivity of an HLA-B27-Specific CTL Clone by Altered Peptide Ligands of Its Natural Epitope
Authors: García-Peydró Marina , Paradela Alberto , Albar Juan P. , Castro José A. López de
Published: April 2014

Antagonism of allospecific CTL by altered MHC ligands is a potential approach to specific immunomodulation of allogeneic T cell responses in acute graft rejection and graft-vs-host disease. In this study we have analyzed the capacity of peptide analogs of a natural HLA-B27-allospecific CTL epitope to antagonize direct alloreactivity. Alanine scanning demonstrated that positions 4, 5, and 7 of the peptide epitope were critical for allorecognition. A number of relatively conservative substitutions at each of these positions were then tested for their effect on allorecognition and antagonism. All substitutions at position 5 abrogated cytotoxicity. In contrast, a few changes at positions 4 and 7 were tolerated, indicating a limited flexibility of the allospecific CTL in recognition of peptide epitope variants. Most of the substitutions impairing cytotoxicity actually induced antagonism. However, whereas epitope variants with changes at positions 4 and 7 behaved as weak or intermediate antagonists, some of the variants with changes at position 5 antagonized CTL alloreactivity almost completely. The results in this study demonstrate for the first time that antagonism of direct class I-mediated alloreactivity can be achieved by variants of a natural allospecific peptide epitope.
Antibodies Directed Against the MHC-I Molecule H-2Dd Complexed with an Antigenic Peptide: Similarities to a T Cell Receptor with the Same Specificity
Authors: Polakova Katarina , Plaksin Daniel , Chung Doo Hyun , Belyakov Igor M. , Berzofsky Jay A. , Margulies David H.
Published: April 2014

αβ TCRs, which use an Ab-like structure to form a combining site, recognize molecular complexes consisting of peptides bound to MHC class I (MHC-I) or class II (MHC-II) molecules. To explore the similarities and differences between Ab and T cell recognition of similar structures, we have isolated two mAbs, KP14 and KP15, that specifically bind H-2Dd complexed with an HIV envelope gp160-derived peptide, P18-I10. These Abs are MHC and peptide specific. Fine specificity of mAb binding was analyzed using a panel of synthetic peptides, revealing similarities between the mAb and a cloned TCR with the same specificity. These two mAbs used the same VH and JH gene segments, but different D, Vκ, and Jκ genes. Administered in vivo, mAb KP15 blocked the induction of CTL specific for recombinant vaccinia virus-encoded gp160, indicating its ability to bind endogenously generated MHC/peptide complexes. Analysis of the fine specificity of these mAbs in the context of their encoded amino acid sequences and the known three-dimensional structure of the H-2Dd/P18-I10 complex suggests that they bind in an orientation similar to that of the TCR. Thus, the plasticity of the B cell receptor repertoire and the structural similarities among BCR and TCR allow Abs to effectively mimic αβ TCRs. Such mAbs may be useful in the therapeutic modulation of immune responses against infectious agents or harmful self Ags as well as in tracing steps in Ag processing.
CTL-Mediated Killing of IntracellularMycobacterium tuberculosisIs Independent of Target Cell Nuclear Apoptosis
Authors: Thoma-Uszynski Sybille , Stenger Steffen , Modlin Robert L.
Published: April 2014

Two subsets of human CTL have been defined based upon phenotype and function: CD4− CD8− double-negative (DN) CTL lyse susceptible targets via Fas-Fas ligand interaction and CD8+ CTL via the granule exocytosis pathway. CD8+ CTL, but not DN CTL, can mediate an antimicrobial activity against Mycobacterium tuberculosis-infected target cells that is dependent on cytotoxic granules that contain granulysin. We investigated the role of nuclear apoptosis for the antimicrobial effector function of CD1-restricted CTL using the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. We found that DN CTL-induced target cell lysis was completely dependent on caspase activation, whereas the cytolytic activity of CD8+ CTL was caspase independent. However, both DN and CD8+ CTL-induced nuclear apoptosis required caspase activation. More important, the antimicrobial effector function of CD8+ CTL was not diminished by inhibition of caspase activity. These data indicate that target cell nuclear apoptosis is not a requirement for CTL-mediated killing of intracellular M. tuberculosis.
Specific Deficiency of p56lck Expression in T Lymphocytes from Type 1 Diabetic Patients
Authors: Nervi Solange , Atlan-Gepner Catherine , Kahn-Perles Brigitte , Lecine Patrick , Vialettes Bernard , Imbert Jean , Naquet Philippe
Published: April 2014

Peripheral T lymphocyte activation in response to TCR/CD3 stimulation is reduced in type 1 diabetic patients. To explore the basis of this deficiency, a comprehensive analysis of the signal transduction pathway downstream of the TCR/CD3 complex was performed for a cohort of patients (n = 38). The main result of the study shows that T cell hyporesponsiveness is positively correlated with a reduced amount of p56lck in resting T lymphocytes. Upon CD3-mediated activation, this defect leads to a hypophosphorylation of the CD3ζ-chain and few other polypeptides without affecting the recruitment of ZAP70. Other downstream effectors of the TCR/CD3 transduction machinery, such as phosphatidylinositol 3-kinase p85α, p59fyn, linker for activation of T cells (LAT), and phospholipase C-γ1, are not affected. In some patients, the severity of this phenotypic deficit could be linked to low levels of p56lck mRNA and resulted in the failure to efficiently induce the expression of the CD69 early activation marker. We propose that a primary deficiency in human type 1 diabetes is a defect in TCR/CD3-mediated T cell activation due to the abnormal expression of the p56lck tyrosine kinase.
Contribution of CD95 Ligand-Induced Neutrophil Infiltration to the Bystander Effect in p53 Gene Therapy for Human Cancer
Authors: Waku Toshihiko , Fujiwara Toshiyoshi , Shao Jianghua , Itoshima Takahiro , Murakami Takayoshi , Kataoka Masafumi , Gomi Shinya , Roth Jack A. , Tanaka Noriaki
Published: April 2014

Clinical trials of adenoviral p53 gene therapy provide the evidence that the bystander effect induced by the wild-type p53 gene transfer on adjacent tumor cells contributes to tumor progression; its mechanism, however, remains uncharacterized. We report in this work that injection of adenovirus expressing the human wild-type p53 gene (Ad5CMVp53) into established human colorectal tumors in nu/nu mice resulted in CD95 ligand (CD95L) overexpression, followed by a massive neutrophil infiltration. Culture supernatants of human colorectal cancer cells infected with Ad5CMVp53 exhibited a potent chemotactic activity against murine polymorphonuclear neutrophils, which could be abolished by the anti-CD95L mAb (NOK-1). In vivo cell depletion experiments indicated that neutrophils were in part responsible for the antitumor effect of the Ad5CMVp53 infection. Our data directly suggest that overexpression of CD95L by the wild-type p53 gene transfer induces neutrophil infiltration into human colorectal tumors, which may play a critical role in the bystander effect of p53 gene therapy.
Neonatal Dexamethasone Treatment Increases Susceptibility to Experimental Autoimmune Disease in Adult Rats
Authors: Bakker Joost M. , Kavelaars Annemieke , Kamphuis Patrick J. G. H. , Cobelens Pieter M. , van Vugt Harmke H. , van Bel Frank , Heijnen Cobi J.
Published: April 2014

Major concern has emerged about the possible long term adverse effects of glucocorticoid treatment, which is frequently used for the prevention of chronic lung disease in preterm infants. Here we show that neonatal glucocorticoid treatment of rats increases the severity (p ≤ 0.01) and incidence (p ≤ 0.01) of the inflammatory autoimmune disease experimental autoimmune encephalomyelitis in adult life. In search of possible mechanisms responsible for the increased susceptibility to experimental autoimmune encephalomyelitis, we investigated the reactivity of the hypothalamo-pituitary-adrenal axis and of immune cells in adult rats after neonatal glucocorticoid treatment. We observed that neonatal glucocorticoid treatment reduces the corticosterone response after an LPS challenge in adult rats (p ≤ 0.001). Interestingly, LPS-stimulated macrophages of glucocorticoid-treated rats produce less TNF-α and IL-1β in adult life than control rats (p < 0.05). In addition, splenocytes obtained from adult rats express increased mRNA levels of the proinflammatory cytokines IFN-γ (p < 0.01) and TNF-β (p < 0.05) after neonatal glucocorticoid treatment. Apparently, neonatal glucocorticoid treatment has permanent programming effects on endocrine as well as immune functioning in adult life. In view of the frequent clinical application of glucocorticoids to preterm infants, our data demonstrate that neonatal glucocorticoid treatment may be a risk factor for the development of (auto)immune disease in man.
Antigen-Specific T Cells Transduced with IL-10 Ameliorate Experimentally Induced Arthritis Without Impairing the Systemic Immune Response to the Antigen
Authors: Setoguchi Keigo , Misaki Yoshikata , Araki Yasuto , Fujio Keishi , Kawahata Kimito , Kitamura Toshio , Yamamoto Kazuhiko
Published: April 2014

For the treatment of rheumatoid arthritis, efficient drug delivery methods to the inflamed joints need to be developed. Because T cells expressing an appropriate autoantigen-specific receptor can migrate to inflamed lesions, it has been reasoned that they can be employed to deliver therapeutic agents. To examine the ability and efficiency of such T cells as a vehicle, we employed an experimentally induced model of arthritis. Splenic T cells from DO11.10 TCR transgenic mice specific for OVA were transduced with murine IL-10. Adoptive transfer of the IL-10-transduced DO11.10 splenocytes ameliorated OVA-induced arthritis despite the presence of around 95% nontransduced cells. Using green fluorescent protein as a marker for selection, the number of transferred cells needed to ameliorate the disease was able to be reduced to 104. Preferential accumulation of the transferred T cells was observed in the inflamed joint, and the improvement in the disease was not accompanied by impairment of the systemic immune response to the Ag, suggesting that the transferred T cells exert their anti-inflammatory task locally, mainly in the joints where the Ag exists. In addition, IL-10-transduced DO11.10 T cells ameliorated methylated BSA-induced arthritis when the arthritic joint was coinjected with OVA in addition to methylated BSA. These results suggest that T cells specific for a joint-specific Ag would be useful as a therapeutic vehicle in rheumatoid arthritis for which the arthritic autoantigen is still unknown.
Cutting Edge: STAT6-Deficient Mice Have Enhanced Tumor Immunity to Primary and Metastatic Mammary Carcinoma
Authors: Ostrand-Rosenberg Suzanne , Grusby Michael J. , Clements Virginia K.
Published: April 2014

STAT4 and STAT6 are essential for the development of CD4+ Th1 and Th2 development, respectively. Tumor immunologists have hypothesized that Th1 cells are critical in tumor immunity because they facilitate differentiation of CD8+ T cells, which are potent anti-tumor effectors. We have used STAT4−/− and STAT6−/− mice to test this hypothesis. BALB/c and knockout mice were challenged in the mammary gland with the highly malignant and spontaneously metastatic BALB/c-derived 4T1 mammary carcinoma. Primary tumor growth and metastatic disease are reduced in STAT6−/− mice relative to BALB/c and STAT4−/− mice. Ab depletions demonstrate that the effect is mediated by CD8+ T cells, and immunized STAT6−/− mice have higher levels of 4T1-specific CTL than BALB/c or STAT4−/− mice. Surprisingly, Th1 or Th2 cells are not involved, because CD4 depletion does not diminish the anti-tumor effect. Therefore, deletion of the STAT6 gene facilitates development of potent anti-tumor immunity via a CD4+-independent pathway.
Spontaneous Rejection of Poorly Immunogenic P1.HTR Tumors by Stat6-Deficient Mice
Authors: Kacha Aalok K. , Fallarino Francesca , Markiewicz Mary A. , Gajewski Thomas F.
Published: April 2014

Experimental evidence suggests that a type 1 T cell response may result in optimal tumor rejection in vivo. This phenotype is determined in part by cytokines that influence T cell differentiation. In transplantable tumor models such as P1.HTR, tumors grow progressively despite expression of defined tumor Ags. We hypothesized that this failure to reject may be due to poor generation of a type 1 phenotype, through a dominant influence of the type 2-promoting cytokines IL-4 and/or IL-13. This hypothesis was tested by implanting P1.HTR tumors into mice deficient in Stat6. In contrast to progressive growth of P1.HTR tumors in wild-type mice, and aggressive growth even of IL-12-transfected P1.HTR in Stat1−/− mice, P1.HTR was spontaneously rejected by Stat6−/− mice. Rejection was accompanied by augmented tumor-specific IFN-γ production and CTL activity. These results suggest that pharmacologic inhibition of Stat6 signaling could potentiate anti-tumor immunity in vivo.
Altered Selection of CD4+ T Cells by Class II MHC Bound with Dominant and Low Abundance Self-Peptides
Authors: Gaszewska-Mastalarz Anna , Muranski Pawel , Chmielowski Bartosz , Kraj Piotr , Ignatowicz Leszek
Published: April 2014

We have investigated the development of CD4+ T cells in mice expressing low levels of transgenic class II MHC molecules (Ab) preoccupied with covalent peptide (Ep), which in the presence of invariant chain (Ii) is extensively cleaved and replaced with self-derived peptides. In these mice, the transgenic Ab molecules, bound with predominant peptide (Ep) and with multiple self-peptides, selected more CD4+ T cells than Ab/self-peptide complexes expressed in wild-type mice. The enhanced outcome of thymic selection was a result of impaired negative selection, rather than more efficient positive selection by an overall lowered abundance of self-derived Ab/peptide complexes. Peripheral CD4+ T cells in the AbEpIi+ mice had memory phenotype, often followed by polyclonal activation of B cells. The AbEpIi+ mice preserved their good health and had a normal life span despite the profound number of activated CD4+ T cells and B cells in peripheral lymphoid organs, moderate hypergammaglobulinemia, and deposited complexes in the kidneys. We propose that CD4+ T cells positively selected due to low avidity for high abundant AbEp complex avoid negative selection on Ab molecules loaded with low abundant peptides and become self-reactive in the peripheral lymphoid organs.
Absence of Functional Inducible NO Synthase Enhances the Efficacy of Tolerance Induced by High Dose Antigen Feeding
Authors: Kahn Daniel A. , Archer D. Clay , Kelly Carolyn J.
Published: April 2014

Recent studies have suggested that IL-12 and IFN-γ may impair the ability of fed Ag to induce systemic tolerance. Because both of these cytokines can function to directly or indirectly induce inducible NO synthase (iNOS) expression, we have investigated whether the functional expression of iNOS regulates oral tolerance. C57BL/6J wild-type or C57BL/6J NOS2−/− mice were gavaged with a single dose of 20 mg of keyhole limpet hemocyanin (KLH), followed by s.c. immunization with KLH/CFA. In the absence of feeding Ag, several parameters of the immune response were more robust in C57BL/6J NOS2−/− mice following KLH/CFA immunization, including the magnitude of the delayed-type hypersensitivity response, the proliferative response, and the production of IFN-γ and IL-2 by Ag-activated draining lymph node cells. These heightened responses in the C57BL/6J NOS2−/− mice are still effectively inhibited by feeding KLH. Feeding KLH to the C57BL/6J NOS2−/− mice elicited heightened TGF-β1 production by Ag-activated lymphocytes, as well as augmented total IgG, IgG1, and IgG2a responses to KLH/CFA compared with that seen in Ag-fed wild-type mice. Feeding Ag to the NOS2−/− mice suppressed proliferative responses and IFN-γ production, while increasing IL-4 production and the IgG1/IgG2a ratio even following a booster immunization of KLH/CFA. Administrating l-N6-(1-iminoethyl)-lysine · 2HCl to wild-type mice during the period of Ag feeding reproduced the high TGF-β1 production seen in Ag-activated lymphocytes from Ag-fed NOS2−/− mice. Feeding KLH is followed by transient up-regulation of NOS2 mRNA expression in the Peyer’s patches of wild-type mice. Selective inhibition of NOS2 may be a simple way to augment tolerogenic mucosal immune responses.
In Vivo Cross-Presentation of a Soluble Protein Antigen: Kinetics, Distribution, and Generation of Effector CTL Recognizing Dominant and Subdominant Epitopes
Authors: Nelson Delia , Bundell Christine , Robinson Bruce
Published: April 2014

Cross-presentation of exogenous Ags via the MHC class I pathway is now recognized for its role in self-tolerance, tumor immunity, and vaccine development. However, little is known about the in vivo distribution and kinetics of cross-presented protein Ags, nor the subsequent development of CTL effector responses to dominant or subdominant epitopes. We examined the location and duration of cross-presented Ag by using 5,6-carboxy-succinimidyl-fluorescein ester-labeled T cells from class I-restricted Ag-specific TCR mice. Comparisons of results from an in vitro 51Cr release CTL assay with an in vivo CTL assay provided physiologically relevant insights into the functional capacities of CTL specific for epitopes with differing affinities. These data demonstrate that efficient cross-presentation of a dominant class I-restricted Ag is dose related and remains largely localized, but not limited to the draining lymph nodes for up to 3 wk following a single injection of soluble protein. Within this period, dominant peptide-specific CTL are fully functional in vivo throughout the secondary lymphoid system. However, no in vivo responses are seen to a subdominant or cryptic epitope. Prolonging Ag cross-presentation via use of IFA promoted persisting in vivo dominant epitope-specific CTL activity and revealed dose-responsive precursor CTL to the subdominant, but not to a cryptic epitope. Analysis of functional in vivo CTL responses demonstrated that, in the presence of strong ongoing responses to the dominant peptide, lytic activity of CTL directed at weaker epitopes is undetectable.
Vaccination with Empty Plasmid DNA or CpG Oligonucleotide Inhibits Diabetes in Nonobese Diabetic Mice: Modulation of Spontaneous 60-kDa Heat Shock Protein Autoimmunity
Authors: Quintana Francisco J. , Rotem Asaf , Carmi Pnina , Cohen Irun R.
Published: April 2014

Nonobese diabetic (NOD) mice develop insulitis and diabetes through a process involving autoimmunity to the 60-kDa heat shock protein (HSP60). Treatment of NOD mice with HSP60 or with peptides derived from HSP60 inhibits this diabetogenic process. We now report that NOD diabetes can be inhibited by vaccination with a DNA construct encoding human HSP60, with the pcDNA3 empty vector, or with an oligonucleotide containing the CpG motif. Prevention of diabetes was associated with a decrease in the degree of insulitis and with down-regulation of spontaneous proliferative T cell responses to HSP60 and its peptide p277. Moreover, both the pcDNA3 vector and the CpG oligonucleotide induced specific Abs, primarily of the IgG2b isotype, to HSP60 and p277, and not to other islet Ags (glutamic acid decarboxylase or insulin) or to an unrelated recombinant Ag expressed in bacteria (GST). The IgG2b isotype of the specific Abs together with the decrease in T cell proliferative responses indicate a shift of the autoimmune process to a Th2 type in treated mice. These results suggest that immunostimulation by bacterial DNA motifs can modulate spontaneous HSP60 autoimmunity and inhibit NOD diabetes.
IL-4-Activated STAT-6 Inhibits IFN-γ-Induced CD40 Gene Expression in Macrophages/Microglia
Authors: Nguyen Vince T. , Benveniste Etty N.
Published: April 2014

The antagonism between the cytokines IFN-γ and IL-4 is well documented, but the mechanism by which IL-4 inhibits IFN-γ-induced gene expression is not clearly understood. CD40 is a type I transmembrane protein that is critical for proper functioning of the immune system. We have previously shown that IFN-γ is the most potent inducer of CD40 expression by macrophages and microglia. In this report, we describe the molecular mechanisms by which IL-4 inhibits IFN-γ-induced CD40 expression. IL-4 suppresses IFN-γ-induced CD40 gene expression in both macrophages and microglia, and such inhibition is dependent on the activation of STAT-6. Nuclear run-on and transfection studies indicate that IL-4-mediated repression is at the transcriptional level. Furthermore, IL-4 inhibition of IFN-γ-induced CD40 expression is specific, since IL-4 does not inhibit IFN-γ-induced IFN-responsive factor-1 gene expression. Site-directed mutagenesis studies demonstrate that two STAT binding sites, named proximal and distal IFN-γ-activated sequences, in the human CD40 promoter are important for IL-4 inhibition of IFN-γ-induced CD40 promoter activity. Moreover, EMSAs indicate that IL-4-activated STAT-6 binds to these two STAT binding sites. These results suggest that IL-4 inhibition of IFN-γ-induced CD40 gene expression is mediated by direct STAT-6 binding to the CD40 promoter.
Enhanced Expression of Fas-Associated Death Domain-Like IL-1-Converting Enzyme (FLICE)-Inhibitory Protein Induces Resistance to Fas-Mediated Apoptosis in Activated Mast Cells
Authors: Yoshikawa Hideshi , Nakajima Yasuo , Tasaka Kachio
Published: April 2014

Mast cells play a critical role in host immune responses and are implicated in the pathogenesis of allergic inflammation. Though mouse mast cell line MC/9 expresses cell surface Fas Ag and is sensitive to Fas-induced apoptosis, activated MC/9 cells are resistant to Fas-induced cell death by cross-linking of FcεRI or FcγR. Fas-associated death domain-like IL-1-converting enzyme (FLICE)-inhibitory protein (FLIP), a caspase-8 inhibitor that lacks the cisteine domain, is one of the negative regulators of receptor-mediated apoptosis. In this report, we show that activation of mast cells by cross-linking of FcεRI or FcγR can induce enhanced expression of FLIP and consequently a resistance to Fas-induced apoptosis, although the expression level of Fas Ag is not changed. Addition of antisense oligonucleotide for FLIP prevents resistance to Fas-induced apoptosis of activated mast cells, suggesting that endogenous FLIP inhibits Fas-mediated apoptosis in activated mast cells. Thus, the enhanced expression of FLIP in activated mast cells contributes to the resistance to Fas-induced apoptosis, which may result in the development and prolongation of allergic inflammation.
Tissue-Specific Expression of Rat IL-18 Gene and Response to Adrenocorticotropic Hormone Treatment
Authors: Sugama Shuei , Kim Yoonseong , Baker Harriet , Tinti Cristina , Kim Hocheol , Joh Tong H. , Conti Bruno
Published: April 2014

IL-18 is a pleiotropic cytokine also proposed to have a role in modulating immune function during stress. Initially found in immune cells, IL-18 mRNA is detectable in several tissues including the cells of the zona reticularis and the zona fasciculata of the adrenal cortex, where its levels are elevated by acute stress or adrenocorticotropic hormone treatment. In the present study, we compared the expression of IL-18 in the adrenal cortex with that of spleen and duodenum, two other IL-18-positive tissues. In situ hybridization showed that, in contrast to the adrenal cortex, in spleen and duodenum IL-18 is primarily localized to immune cells. In duodenum, IL-18 mRNA was also detectable in epithelial cells. Northern blot demonstrated that while the adrenal gland synthesized IL-18 mRNA of 1.1 kb, spleen and duodenum produced a 0.9-kb transcript. RT-PCR, sequencing, Western blot, primer extension, and rapid amplification of cDNA end analysis demonstrated that the three tissues synthesize IL-18 mRNAs containing the same coding region and produce the same IL-18 peptide, but differ in the length of their 5′-untranslated region, indicating tissue-specific usage of the promoter region. Finally, in contrast to the adrenal gland, adrenocorticotropic hormone treatment did not increase the levels of IL-18 mRNA in spleen and duodenum. These results demonstrate tissue-specific expression and promoter usage of IL-18 gene and suggest that the adrenal cortex and not immune cells may be the source of IL-18 during stress.