bluuebunny/crossref_metadata_2025 huggingface dataset:
Antiretroviral Drug Resistance Mutations Sustain or Enhance CTL Recognition of Common HIV-1 Pol Epitopes
Authors: Mason Rosemarie D. , Bowmer M. Ian , Howley Constance M. , Gallant Maureen , Myers Jennifer C. E. , Grant Michael D.
Published: April 2014

Antiretroviral drug resistance and escape from CTL are major obstacles to effective control of HIV replication. To investigate the possibility of combining drug and immune-based selective pressures against HIV, we studied the effects of antiretroviral drug resistance mutations on CTL recognition of five HIV-1 Pol epitopes presented by common HLA molecules. We found that these common drug resistance mutations sustain or even enhance the antigenicity and immunogenicity of HIV-1 Pol CTL epitopes. Variable patterns of cross-reactive and selective recognition of wild-type and corresponding variant epitopes demonstrate a relatively diverse population of CD8+ T cells reactive against these epitopes. Variant peptides with multiple drug resistance mutations still sustained CTL recognition, and some HIV-infected individuals demonstrated strong CD8+ T cell responses against multiple CTL epitopes incorporating drug resistance mutations. Selective reactivity against variant peptides with drug resistance mutations reflected ongoing or previous exposure to the indicated drug, but was not dependent upon the predominance of the mutated sequence in endogenous virus. The frequency and diversity of CTL reactivity against the variant peptides incorporating drug resistance mutations and the ability of these peptides to activate and expand CTL precursors in vitro indicate a significant functional interface between the immune system and antiretroviral therapy. Thus, drug-resistant variants of HIV are susceptible to immune selective pressure that could be applied to combat transmission or emergence of antiretroviral drug-resistant HIV strains and to enhance the immune response against HIV.
Quantitative Analysis of Prion-Protein Degradation by Constitutive and Immuno-20S Proteasomes Indicates Differences Correlated with Disease Susceptibility
Authors: Tenzer Stefan , Stoltze Lars , Schönfisch Birgitt , Dengjel Jörn , Müller Margret , Stevanović Stefan , Rammensee Hans-Georg , Schild Hansjörg
Published: April 2014

The main part of cytosolic protein degradation depends on the ubiquitin-proteasome system. Proteasomes degrade their substrates into small peptide fragments, some of which are translocated into the endoplasmatic reticulum and loaded onto MHC class I molecules, which are then transported to the cell surface for inspection by CTL. A reliable prediction of proteasomal cleavages in a given protein for the identification of CTL epitopes would benefit immensely from additional cleavage data for the training of prediction algorithms. To increase the knowledge about proteasomal specificity and to gain more insight into the relation of proteasomal activity and susceptibility to prion disease, we digested sheep prion protein with human constitutive and immuno-20S proteasomes. All fragments generated in the digest were quantified. Our results underline the different cleavage specificities of constitutive and immunoproteasomes and provide data for the training of prediction programs for proteasomal cleavages. Furthermore, the kinetic analysis of proteasomal digestion of two different alleles of prion protein shows that even small changes in a protein sequence can affect the overall efficiency of proteasomal processing and thus provides more insight into the possible molecular background of allelic variations and the pathogenicity of prion proteins.
Enhanced Catalytic Action of HLA-DM on the Exchange of Peptides Lacking Backbone Hydrogen Bonds between their N-Terminal Region and the MHC Class II α-Chain
Authors: Stratikos Efstratios , Wiley Don C. , Stern Lawrence J.
Published: April 2014

The class II MHC homolog HLA-DM catalyzes exchange of peptides bound to class II MHC proteins, and is an important component of the Ag presentation machinery. The mechanism of HLA-DM-mediated catalysis is largely obscure. HLA-DM catalyzes exchange of peptides of varying sequence, suggesting that a peptide sequence-independent component of the MHC-peptide interaction could be involved in the catalytic process. Twelve conserved hydrogen bonds between the peptide backbone and the MHC are a prominent sequence-independent feature of the MHC-peptide interaction. To evaluate the relative importance of these hydrogen bonds toward HLA-DM action, we prepared peptide variants that lacked the ability to form one or more of the hydrogen bonds as a result of backbone amide N-methylation or truncation, and tested their ability to be exchanged by HLA-DM. We found that disruption of hydrogen bonds involving HLA-DR1 residues α51–53, a short extended segment at the N terminus of the α subunit helical region, led to heightened HLA-DM catalytic efficacy. We propose that those bonds are disrupted in the MHC conformation recognized by HLA-DM to allow structural transitions in that area during DM-assisted peptide release. These results suggest that peptides or compounds that bind MHC but cannot form these interactions would be preferentially edited out by HLA-DM.
Staphylococcus aureus Resists Human Defensins by Production of Staphylokinase, a Novel Bacterial Evasion Mechanism
Authors: Jin Tao , Bokarewa Maria , Foster Timothy , Mitchell Jennifer , Higgins Judy , Tarkowski Andrej
Published: April 2014

α-Defensins are peptides secreted by polymorphonuclear cells and provide antimicrobial protection mediated by disruption of the integrity of bacterial cell walls. Staphylokinase is an exoprotein produced by Staphylococcus aureus, which activates host plasminogen. In this study, we analyzed the impact of interaction between α-defensins and staphylokinase on staphylococcal growth. We observed that staphylokinase induced extracellular release of α-defensins from polymorphonuclear cells. Moreover, a direct binding between α-defensins and staphylokinase was shown to result in a complex formation. The biological consequence of this interaction was an almost complete inhibition of the bactericidal effect of α-defensins. Notably, staphylokinase with blocked plasminogen binding site still retained its ability to neutralize the bactericidal effect of α-defensins. In contrast, a single mutation of a staphylokinase molecule at position 74, substituting lysine for alanine, resulted in a 50% reduction of its α-defensin-neutralizing properties. The bactericidal properties of α-defensins were tested in 19 S. aureus strains in vitro and in a murine model of S. aureus arthritis. Staphylococcal strains producing staphylokinase were protected against the bactericidal effect of α-defensins. When staphylokinase was added to staphylokinase-negative S. aureus cultures, it almost totally abrogated the effect of α-defensins. Finally, human neutrophil peptide 2 injected intra-articularly along with bacteria alleviated joint destruction. In this study, we report a new property of staphylokinase, its ability to induce secretion of defensins, to complex bind them and to neutralize their bactericidal effect. Staphylokinase production may therefore be responsible in vivo for defensin resistance during S. aureus infections.
Expression of Signaling Lymphocytic Activation Molecule- Associated Protein Interrupts IFN-γ Production in Human Tuberculosis
Authors: Pasquinelli Virginia , Quiroga María F. , Martínez Gustavo J. , Zorrilla Liliana Castro , Musella Rosa M. , Bracco María M. , Belmonte Liliana , Malbrán Alejandro , Fainboim Leonardo , Sieling Peter A. , García Verónica E.
Published: April 2014

Production of the Th1 cytokine IFN-γ by T cells is considered crucial for immunity against Mycobacterium tuberculosis infection. We evaluated IFN-γ production in tuberculosis in the context of signaling molecules known to regulate Th1 cytokines. Two populations of patients who have active tuberculosis were identified, based on their T cell responses to the bacterium. High responder tuberculosis patients displayed significant M. tuberculosis-dependent T cell proliferation and IFN-γ production, whereas low responder tuberculosis patients displayed weak or no T cell responses to M. tuberculosis. The expression of the signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) on cells from tuberculosis patients was inversely correlated with IFN-γ production in those individuals. Moreover, patients with a nonfunctional SAP gene displayed immune responses to M. tuberculosis similar to those of high responder tuberculosis patients. In contrast to SAP, T cell expression of SLAM was directly correlated with responsiveness to M. tuberculosis Ag. Our data suggest that expression of SAP interferes with Th1 responses whereas SLAM expression contributes to Th1 cytokine responses in tuberculosis. The study further suggests that SAP and SLAM might be focal points for therapeutic modulation of T cell cytokine responses in tuberculosis.
Generation of a Complement-Independent Bactericidal IgM against a Relapsing Fever Borrelia
Authors: Connolly Sean E. , Thanassi David G. , Benach Jorge L.
Published: April 2014

The spirochetemia of relapsing fever in mice is cleared by a complement-independent, polyclonal IgM response with reactivity to two prominent Ags of 20 and 35 kDa. In this study, we have dissected the polyclonal IgM Ab response against a relapsing fever spirochete to determine the specificity of its complement-independent bactericidal properties. Our experimental approach selectively generated an IgM murine mAb from the early specific immune response to a variable outer membrane protein. This IgM is bactericidal in the absence of complement and is part of the polyclonal Ab response that mediates the clearance of this bacterium from the blood. Purified monoclonal IgM caused direct structural damage to the outer membrane of the spirochete, in the absence of complement, and protected both B cell- and C5-deficient mice from challenge when administered passively. The direct, complement-independent, bactericidal activity of Abs is a critical mechanism of host defense against infection.
L-Ficolin Specifically Binds to Lipoteichoic Acid, a Cell Wall Constituent of Gram-Positive Bacteria, and Activates the Lectin Pathway of Complement
Authors: Lynch Nicholas J. , Roscher Silke , Hartung Thomas , Morath Siegfried , Matsushita Misao , Maennel Daniela N. , Kuraya Mikio , Fujita Teizo , Schwaeble Wilhelm J.
Published: April 2014

The lectin pathway of complement is activated when a carbohydrate recognition complex and associated serine proteases binds to the surface of a pathogen. Three recognition subcomponents have been shown to form active initiation complexes: mannan-binding lectin (MBL), L-ficolin, and H-ficolin. The importance of MBL in antimicrobial host defense is well recognized, but the role of the ficolins remains largely undefined. This report shows that L-ficolin specifically binds to lipoteichoic acid (LTA), a cell wall component found in all Gram-positive bacteria. Immobilized LTA from Staphylococcus aureus binds L-ficolin complexes from sera, and these complexes initiate lectin pathway-dependent C4 turnover. C4 activation correlates with serum L-ficolin concentration, but not with serum MBL levels. L-ficolin binding and corresponding levels of C4 turnover were observed on LTA purified from other clinically important bacteria, including Streptococcus pyogenes and Streptococcus agalactiae. None of the LTA preparations bound MBL, H-ficolin, or the classical pathway recognition molecule, C1q.
Different Functional Capacities of Latent and Lytic Antigen-Specific CD8 T Cells in Murine Gammaherpesvirus Infection
Authors: Obar Joshua J. , Crist Sarah G. , Gondek David C. , Usherwood Edward J.
Published: April 2014

Gammaherpesviruses can persist in the host in the face of an aggressive immune response. T cells recognize Ags expressed in both the productive and latent phases of the virus life cycle, however little is known about their relative roles in the long-term control of the infection. In this study we used the murine gammaherpesvirus 68 model system to investigate the relative properties of CD8 T cells recognizing lytic and latent viral Ags. We report that the CD8 T cell response to lytic phase epitopes is maximal in the lungs of infected mice at ∼10 days postinfection, and is of progressively lesser magnitude in the mediastinal lymph nodes and spleen. In contrast, the CD8 T cell response to the latent M2 protein is maximal at ∼19 days postinfection and is most prominent in the spleen, then progressively less in the mediastinal lymph node and the lung. Latent and lytic Ag-specific CD8 T cells had markedly different cell surface phenotypes during chronic infection, with latent Ag-specific cells being predominantly CD62Lhigh or CD43 (1B11)high. Lytic Ag-specific T cells had significantly lower expression of these markers. Importantly, latent but not lytic Ag-specific T cells could kill target cells rapidly in vivo during the chronic infection. These two different sets of CD8 T cells also responded differentially to IL-7, a cytokine involved in T cell homeostasis and the maintenance of T cell memory. These data have important implications for our understanding of immunological control during chronic gammaherpesvirus infections.
Carbon Monoxide Inhibits T Lymphocyte Proliferation via Caspase-Dependent Pathway
Authors: Song Ruiping , Mahidhara Raja S. , Zhou Zhihong , Hoffman Rosemary A. , Seol Dai-Wu , Flavell Richard A. , Billiar Timothy R. , Otterbein Leo E. , Choi Augustine M. K.
Published: April 2014

T lymphocyte activation and proliferation is involved in many pathological processes. We have recently shown that carbon monoxide (CO), an enzymatic product of heme oxyenase-1 (HO-1), confers potent antiproliferative effects in airway and vascular smooth muscle cells. The purpose of this study was to determine whether CO can inhibit T lymphocyte proliferation and then to determine the mechanism by which CO can modulate T lymphocyte proliferation. In the presence of 250 parts per million CO, CD3-activated T lymphocyte proliferation was, remarkably, inhibited by 80% when compared with controls. We observed that the antiproliferative effect of CO in T lymphocytes was independent of the mitogen-activated protein kinase or cGMP signaling pathways, unlike what we demonstrated previously in smooth muscle cells. We demonstrate that CO inhibited caspase-3 and caspase-8 expression and activity, and caspase inhibition with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK pan-caspase inhibitor) blocked T lymphocyte proliferation. Furthermore, in caspase-8-deficient lymphocytes, the antiproliferative effect of CO was markedly attenuated, further supporting the involvement of caspase-8 in the antiproliferative effects of CO. CO also increased the protein level of p21Cip1, and CO-mediated inhibition of caspase activity is partially regulated by p21Cip1. Taken together, these data suggest that CO confers potent antiproliferative effects in CD3-activated T lymphocytes and that these antiproliferative effects in T lymphocytes are mediated by p21Cip1-dependent caspase activity, in particular caspase-8, independent of cGMP and mitogen-activated protein kinase signaling pathways.
Semaphorin CD100 from Activated T Lymphocytes Induces Process Extension Collapse in Oligodendrocytes and Death of Immature Neural Cells
Authors: Giraudon Pascale , Vincent Peggy , Vuaillat Carine , Verlaeten Olivier , Cartier Luis , Marie-Cardine Anne , Mutin Mireille , Bensussan Armand , Belin Marie-Françoise , Boumsell Laurence
Published: April 2014

An inappropriate cross talk between activated T lymphocytes infiltrating the CNS and neural cells can sustain the onset and progression of demyelination and axonal degeneration in neuroinflammatory diseases. To mimic this deleterious cross talk, we designed an experimental paradigm consisting of transient cocultures of T lymphocytes chronically activated by retrovirus infection (not virus productive) with human multipotent neural precursors or primary oligodendrocytes from rat brain. We showed that activated T lymphocytes induced apoptotic death of multipotent neural progenitors and immature oligodendrocytes after a progressive collapse of their process extensions. These effects were reminiscent of those induced by brain semaphorin on neural cells. Blockade by specific Abs of soluble CD100 (sCD100)/semaphorin 4D released by activated T cells, or treatment with rsCD100, demonstrated that this immune semaphorin has the ability to collapse oligodendrocyte process extensions and to trigger neural cell apoptosis, most likely through receptors of the plexin family. The specific presence of sCD100 in the cerebrospinal fluid and of CD100-expressing T lymphocytes in the spinal cord of patients suffering with neuroinflammatory demyelination pointed to the potential pathological effect of sCD100 in the CNS. Thus, our results show that CD100 is a new important element in the deleterious T cell-neural cell cross talk during neuroinflammation and suggest its role in demyelination or absence of remyelination in neuroinflammatory diseases including multiple sclerosis and human T lymphotropic virus type 1-associated myelopathy.
Chemokine Secretion of Rheumatoid Arthritis Synovial Fibroblasts Stimulated by Toll-Like Receptor 2 Ligands
Authors: Pierer Matthias , Rethage Janine , Seibl Reinhart , Lauener Roger , Brentano Fabia , Wagner Ulf , Hantzschel Holm , Michel Beat A. , Gay Renate E. , Gay Steffen , Kyburz Diego
Published: April 2014

To analyze the role of Toll-like receptors (TLR) in the pathogenesis of rheumatoid arthritis, we have assessed the effects of stimulation of cultured synovial fibroblasts by the TLR-2 ligand bacterial peptidoglycan. By using high density oligonucleotide microarray analysis we identified 74 genes that were up-regulated >2.5-fold. Fourteen CC and CXC chemokine genes were among the genes with the highest up-regulation. Quantitative real-time PCR analysis confirmed up-regulation of granulocyte chemotactic protein (GCP)-2, RANTES, monocyte chemoattractant protein (MCP)-2, IL-8, growth-related oncogene-2, and to a lesser extent, macrophage-inflammatory protein 1α, MCP-1, EXODUS, and CXCL-16. GCP-2, RANTES, and MCP-2 were detected in culture supernatants of synovial fibroblasts stimulated with peptidoglycan. Chemokine secretion induced by stimulation of rheumatoid arthritis synovial fibroblasts via TLR-2 was functionally relevant as demonstrated by chemotaxis assays. GCP-2 and MCP-2 expression, which have not been reported previously in rheumatoid arthritis, was demonstrated in synovial tissue sections of patients diagnosed with rheumatoid arthritis but not in those with osteoarthritis. Correspondingly, synovial fluid levels were significantly higher in patients diagnosed with rheumatoid arthritis as compared with osteoarthritis. Thus, we present evidence for an induction of chemokine secretion by activation of synovial fibroblasts via TLR-2, possibly contributing to the formation of inflammatory infiltrates characteristically found in rheumatoid arthritis joints.
Functional Analysis of Tumor-Specific Th Cell Responses Detected in Melanoma Patients after Dendritic Cell-Based Immunotherapy
Authors: Schultz Erwin S. , Schuler-Thurner Beatrice , Stroobant Vincent , Jenne Lars , Berger Thomas G. , Thielemanns Kris , van der Bruggen Pierre , Schuler Gerold
Published: April 2014

Recently, we have demonstrated that tumor-specific CD4+ Th cell responses can be rapidly induced in advanced melanoma patients by vaccination with peptide-loaded monocyte-derived dendritic cells. Most patients showed a T cell reactivity against a melanoma Ag 3 (MAGE-3) peptide (MAGE-3243–258), which has been previously found to be presented by HLA-DP4 molecules. To analyze the functional and specificity profile of this in vivo T cell response in detail, peptide-specific CD4+ T cell clones were established from postvaccination blood samples of two HLA-DP4 patients. These T cell clones recognized not only peptide-loaded stimulator cells but also dendritic cells loaded with a recombinant MAGE-3 protein, demonstrating that these T cells were directed against a naturally processed MAGE-3 epitope. The isolated CD4+ Th cells showed a typical Th1 cytokine profile upon stimulation. From the first patient several CD4+ T cell clones recognizing the antigenic peptide used for vaccination in the context of HLA-DP4 were obtained, whereas we have isolated from the second patient CD4+ T cell clones which were restricted by HLA-DQB1*0604. Analyzing a panel of truncated peptides revealed that the CD4+ T cell clones recognized different core epitopes within the original peptide used for vaccination. Importantly, a DP4-restricted T cell clone was stimulated by dendritic cells loaded with apoptotic or necrotic tumor cells and even directly recognized HLA class II- and MAGE-3-expressing tumor cells. Moreover, these T cells exhibited cytolytic activity involving Fas-Fas ligand interactions. These findings support that vaccination-induced CD4+ Th cells might play an important functional role in antitumor immunity.
Cutting Edge: Anthrax Lethal Toxin Inhibits Activation of IFN-Regulatory Factor 3 by Lipopolysaccharide
Authors: Dang Oanh , Navarro Lorena , Anderson Keith , David Michael
Published: April 2014

IFN-regulatory factor 3 (IRF3) is known to participate in the transcriptional induction of chemokines and cytokines, including IFNs, as a result of viral or bacterial infection. In this study, we demonstrate that the LPS-mediated activation of IRF3 and subsequent induction of chemokine genes or IRF3-responsive reporter constructs are inhibited after exposure of human or murine macrophages to the Bacillus anthracis toxin lethal factor. The inhibitory effect is caused by interference with the activation of the stress-activated protein kinase, p38, due to a proteolytic cleavage of mitogen-activated protein kinase kinase 6, and can be overcome by the ectopic expression of a cleavage-resistant mutant of mitogen-activated protein kinase kinase 6 or a constitutively active IRF3. The lethal factor-mediated inhibition of IRF3 activation and subsequent cytokine production through bacterial membrane components offers Bacillus anthracis an efficient mechanism to evade the innate immune response.
Differential Role of CCR2 in Islet and Heart Allograft Rejection: Tissue Specificity of Chemokine/Chemokine Receptor Function In Vivo
Authors: Abdi Reza , Means Terry K. , Ito Toshiro , Smith Rex Neal , Najafian Nader , Jurewicz Mollie , Tchipachvili Vaja , Charo Israel , Auchincloss Hugh , Sayegh Mohamed H. , Luster Andrew D.
Published: April 2014

Chemokines have a pivotal role in the mobilization and activation of specific leukocyte subsets in acute allograft rejection. However, the role of specific chemokines and chemokine receptors in islet allograft rejection has not been fully elucidated. We now show that islet allograft rejection is associated with a steady increase in intragraft expression of the chemokines CCL8 (monocyte chemoattractant protein-2), CCL9 (monocyte chemoattractant protein-5), CCL5 (RANTES), CXCL-10 (IFN-γ-inducible protein-10), and CXCL9 (monokine induced by IFN-γ) and their corresponding chemokine receptors CCR2, CCR5, CCR1, and CXCR3. Because CCR2 was found to be highly induced, we tested the specific role of CCR2 in islet allograft rejection by transplanting fully MHC mismatched islets from BALB/c mice into C57BL/6 wild-type (WT) and CCR2-deficient mice (CCR2−/−). A significant prolongation of islet allograft survival was noted in CCR2−/− recipients, with median survival time of 24 and 12 days for CCR2−/− and WT recipients, respectively (p < 0.0001). This was associated with reduction in the generation of CD8+, but not CD4+ effector alloreactive T cells (CD62LlowCD44high) in CCR2−/− compared with WT recipients. In addition, CCR2−/− recipients had a reduced Th1 and increased Th2 alloresponse in the periphery (by ELISPOT analysis) as well as in the grafts (by RT-PCR). However, these changes were only transient in CCR2−/− recipients that ultimately rejected their grafts. Furthermore, in contrast to the islet transplants, CCR2 deficiency offered only marginal prolongation of heart allograft survival. This study demonstrates the important role for CCR2 in early islet allograft rejection and highlights the tissue specificity of the chemokine/chemokine receptor system in vivo in regulating allograft rejection.
Marginal Zone, but Not Follicular B Cells, Are Potent Activators of Naive CD4 T Cells
Authors: Attanavanich Kalaya , Kearney John F.
Published: April 2014

The early involvement of marginal zone (MZ) B lymphocytes in T-independent immune responses is well established. In this study we compared the abilities of MZ and follicular (FO) B cells to collaborate with T cells. After immunization with soluble hen egg lysozyme, both MZ and FO B cells captured Ag and migrated to T cell areas in the response to hen egg lysozyme. MZ B cells were far superior to FO B cells in inducing CD4+ T cell expansion both in vitro and in vivo. MZ, but not FO, B cells, after interaction with T cells, differentiated into plasma cells, and in addition they stimulated Ag-specific CD4+ T cells to produce high levels of Th1-like cytokines upon primary stimulation in vitro. These results indicate that MZ B cells rapidly and effectively capture soluble Ag and activate CD4+ T cells to become effector T cells. The enhanced capacity of MZ B cells to prime T cells in this study appeared to be intrinsic to MZ B cells, as both MZ and FO B cell populations express an identical Ag receptor.
TGF-β1 Plays an Important Role in the Mechanism of CD4+CD25+ Regulatory T Cell Activity in Both Humans and Mice
Authors: Nakamura Kazuhiko , Kitani Atsushi , Fuss Ivan , Pedersen Aasta , Harada Naohiko , Nawata Hajime , Strober Warren
Published: April 2014

In previous studies, we have shown that murine CD4+CD25+ regulatory T cells produce high levels of TGF-β1 in a cell surface and/or secreted form, and blockade of such TGF-β1 by anti-TGF-β curtails the ability of these cells to suppress CD25− T cell proliferation and B cell Ig production in in vitro suppressor assays. In further support for the role of TGF-β1 in suppression by CD4+CD25+ T cells, we show in this study that another TGF-β1-blocking molecule, recombinant latency-associated peptide of TGF-β1 (rLAP), also reverses suppression by mouse CD4+CD25+ T cells as well as their human counterparts, CD4+CD25high T cells. In addition, we show that CD25− T cells exposed to CD4+CD25+ T cells in vitro manifest activation of Smad-2 and induction of CD103, the latter a TGF-β-inducible surface integrin. In further studies, we show that while CD4+CD25+ T cells from TGF-β1-deficient mice can suppress CD25− T cell proliferation in vitro, these cells do not protect recipient mice from colitis in the SCID transfer model in vivo, and, in addition, CD4+LAP+, but not CD4+LAP− T cells from normal mice protect recipient mice from colitis in this model. Together, these studies demonstrate that TGF-β1 produced by CD4+CD25+ T cells is involved in the suppressor activity of these cells, particularly in their ability to regulate intestinal inflammation.
Ephrin-A1 Suppresses Th2 Cell Activation and Provides a Regulatory Link to Lung Epithelial Cells
Authors: Wohlfahrt Jan G. , Karagiannidis Christian , Kunzmann Steffen , Epstein Michelle M. , Kempf Werner , Blaser Kurt , Schmidt-Weber Carsten B.
Published: April 2014

Gene expression screening showed decreased ephrin-A1 expression in CD4+ T cells of asthma patients. Ephrin-A1 is the ligand of the Eph receptor family of tyrosine kinases, forming the largest family of receptor tyrosine kinases. Their immune regulatory properties are largely unknown. This study demonstrates significantly reduced ephrin-A1 expression in T cells of asthma patients using real time-PCR. Immunohistological analyses revealed strong ephrin-A1 expression in lung tissue and low expression in cortical areas of lymph nodes. It is absent in T cell/B cell areas of the spleen. Colocalization of ephrin-A1 and its receptors was found only in the lung, but not in lymphoid tissues. In vitro activation of T cells reduced ephrin-A1 at mRNA and protein levels. T cell proliferation, activation-induced, and IL-2-dependent cell death were inhibited by cross-linking ephrin-A1, and not by engagement of Eph receptors. However, anti-EphA1 receptor slightly enhances Ag-specific and polyclonal proliferation of PBMC cultures. Furthermore, activation-induced CD25 up-regulation was diminished by ephrin-A1 engagement. Ephrin-A1 engagement reduced IL-2 expression by 82% and IL-4 reduced it by 69%; the IFN-γ expression remained unaffected. These results demonstrate that ephrin-A1 suppresses T cell activation and Th2 cytokine expression, while preventing activation-induced cell death. The reduced ephrin-A1 expression in asthma patients may reflect the increased frequency of activated T cells in peripheral blood. That the natural ligands of ephrin-A1 are most abundantly expressed in the lung may be relevant for Th2 cell regulation in asthma and Th2 cell generation by mucosal allergens.
Dissociation of NKT Stimulation, Cytokine Induction, and NK Activation In Vivo by the Use of Distinct TCR-Binding Ceramides
Authors: Ortaldo John R. , Young Howard A. , Winkler-Pickett Robin T. , Bere Earl W. , Murphy William J. , Wiltrout Robert H.
Published: April 2014

NKT and NK cells are important immune regulatory cells. The only efficient means to selectively stimulate NKT cells in vivo is α-galactosylceramide (αGalCer). However, αGalCer effectively stimulates and then diminishes the number of detectable NKT cells. It also exhibits a potent, indirect ability to activate NK cells. We have now discovered another ceramide compound, β-galactosylceramide (βGalCer) (C12), that efficiently diminishes the number of detectable mouse NKT cells in vivo without inducing significant cytokine expression or activation of NK cells. Binding studies using CD1d tetramers loaded with βGalCer (C12) demonstrated significant but lower intensity binding to NKT cells when compared with αGalCer, but both ceramides were equally efficient in reducing the number of NKT cells. However, βGalCer (C12), in contrast to αGalCer, failed to increase NK cell size, number, and cytolytic activity. Also in contrast to αGalCer, βGalCer (C12) is a poor inducer of IFN-γ, TNF-α, GM-CSF, and IL-4 gene expression. These qualitative differences in NKT perturbation/NK activation have important implications for delineating the unique in vivo roles of NKT vs NK cells. Thus, αGalCer (which triggers NKT cells and activates NK cells) efficiently increases the resistance to allogeneic bone marrow transplantation while βGalCer (C12) (which triggers NKT cells but does not activate NK cells) fails to enhance bone marrow graft rejection. Our results show βGalCer (C12) can effectively discriminate between NKT- and NK-mediated responses in vivo. These results indicate the use of different TCR-binding ceramides can provide a unique approach for understanding the intricate immunoregulatory contributions of these two cell types.
The Cell Surface-Localized Heat Shock Protein 70 Epitope TKD Induces Migration and Cytolytic Activity Selectively in Human NK Cells
Authors: Gastpar Robert , Gross Catharina , Rossbacher Lydia , Ellwart Joachim , Riegger Julia , Multhoff Gabriele
Published: April 2014

Profiling of surface-bound proteins uncovers a tumor-selective heat shock protein 70 (Hsp70) membrane expression that provides a target structure for human NK cells. Hsp70 peptide TKD (TKDNNLLGRFELSG; aa 450–463) was found to enhance the cytolytic activity of NK cells. In this study, we demonstrate that TKD-activated CD3−CD56+CD94+ NK cells are selectively attracted by Hsp70 membrane-positive tumor cells, and supernatants derived thereof. Hsp70 membrane-negative tumors failed to attract these NK cells. The capacity to migrate was associated with a substantial lytic activity against Hsp70-positive tumor cells. Because NK cell migration was independent of cell-to-cell contact, the involvement of a soluble factor was assumed. Interestingly, synthetic Hsp70 protein and Hsp70 peptide TKD, mimicking surface-bound Hsp70, initiates migration of NK cells in a concentration-dependent (1–5 μg/ml), highly selective, and chemokine-independent manner. In summary, our results indicate that Hsp70 peptide TKD not only stimulates cytolysis but also chemotaxis in CD3−CD56+CD94+ NK cells.
Mechanistic Insights into Impaired Dendritic Cell Function by Rapamycin: Inhibition of Jak2/Stat4 Signaling Pathway
Authors: Chiang Po-Hui , Wang Lianfu , Bonham C. Andrew , Liang Xiaoyan , Fung John J. , Lu Lina , Qian Shiguang
Published: April 2014

The suppressive effect of rapamycin on T cells has been extensively studied, but its influence on the function of APC is less clear. The data in this study demonstrated that immunostimulatory activity of B10 (H2b) dendritic cells (DC) exposed to rapamycin (rapa-DC) was markedly suppressed as evidenced by the induction of low proliferative responses and specific CTL activity in allogeneic (C3H, H2k) T cells. Administration of rapa-DC significantly prolonged survival of B10 cardiac allografts in C3H recipients. Treatment with rapamycin did not affect DC expression of MHC class II and costimulatory molecules or IL-12 production. Rapamycin did not inhibit DC NF-κB pathway, however, IL-12 signaling through Janus kinase 2/Stat4 activation was markedly suppressed. Indeed, Stat4−/− DC similarly displayed poor allostimulatory activity. The Stat4 downstream product, IFN-γ, was also inhibited by rapamycin, but DC dysfunction could not solely be attributed to low IFN-γ production as DC deficient in IFN-γ still exhibited vigorous allostimulatory activity. Rapamycin did not affect DC IL-12R expression, but markedly suppressed IL-18Rα and β expression, which may in turn down-regulate DC IL-12 autocrine activation.
Tolerance Induction by Intrathymic Expression of P0
Authors: Visan Lucian , Visan Ioana A. , Weishaupt Andreas , Hofstetter Harald H. , Toyka Klaus V. , Hünig Thomas , Gold Ralf
Published: April 2014

Genetic deficiency or instability of myelin protein zero (P0) results in hereditary motor sensory neuropathy. In view of recent advances in gene therapy, substitution of the molecular defect may become realistic in the near future. Here we investigate the impact of genetic deficiency of P0 on selection of the autoreactive T cell repertoire in the corresponding mouse model. We show that P0 mRNA transcripts are expressed in thymic stroma, similar to other myelin proteins and that expression of intact P0 protein can be detected by Western blot. Using a library of overlapping 20mer peptides spanning the entire length of P0 and applying the ELISPOT technique, we detected a strong immune response toward P0 extracellular domain peptide aa 41–60 in P0−/− knockout mice, but not in heterozygous P0+/− or wild-type (wt) mice. In addition, one cryptic epitope and two subdominant epitopes of P0 were identified. Using P0−/− into wt bone marrow (BM) chimeras we found that P0 expression in the host suffices for full tolerance induction, which is in line with its presence in thymic stroma. However, repopulation of P0−/− mice with wt BM led to partial induction of tolerance, suggesting that BM derived cells can also express this protein. Our findings may have implications for secondary autoimmunity developing after gene therapy in hereditary neuropathies and other diseases with genetically determined protein deficiency, because the repaired protein will then represent a foreign, nontolerized Ag.
Cellular Maturation Defects in Bruton’s Tyrosine Kinase-Deficient Immature B Cells Are Amplified by Premature B Cell Receptor Expression and Reduced by Receptor Editing
Authors: Middendorp Sabine , Hendriks Rudolf W.
Published: April 2014

In the mouse, Bruton’s tyrosine kinase (Btk) is essential for efficient developmental progression of CD43+CD2− large cycling into CD43−CD2+ small resting pre-B cells in the bone marrow and of IgMhigh transitional type 2 B cells into IgMlow mature B cells in the spleen. In this study, we show that the impaired induction of cell surface changes in Btk-deficient pre-B cells was still noticeable in κ+ immature B cells, but was largely corrected in λ+ immature B cells. As λ gene rearrangements are programmed to follow κ rearrangements and λ expression is associated with receptor editing, we hypothesized that the transit time through the pre-B cell compartment or receptor editing may affect the extent of the cellular maturation defects in Btk-deficient B cells. To address this issue, we used 3-83μδ transgenic mice, which prematurely express a complete B cell receptor and therefore manifest accelerated B cell development. In Btk-deficient 3-83μδ mice, the IgM+ B cells in the bone marrow exhibited a very immature phenotype (pre-BCR+CD43+CD2−) and were arrested at the transitional type 1 B cell stage upon arrival in the spleen. However, these cellular maturation defects were largely restored when Btk-deficient 3-83μδ B cells were on a centrally deleting background and therefore targeted for receptor editing. Providing an extended time window for developing B cells by enforced expression of the antiapoptotic gene Bcl-2 did not alter the Btk dependence of their cellular maturation. We conclude that premature B cell receptor expression amplifies the cellular maturation defects in Btk-deficient B cells, while extensive receptor editing reduces these defects.
IL-11 Protects Human Microvascular Endothelium from Alloinjury In Vivo by Induction of Survivin Expression
Authors: Kirkiles-Smith Nancy C. , Mahboubi Keyvan , Plescia Janet , McNiff Jennifer M. , Karras James , Schechner Jeffrey S. , Altieri Dario C. , Pober Jordan S.
Published: April 2014

IL-11 can reduce tissue injury in animal models of inflammation but the mechanism(s) is unknown. When C.B-17 SCID/beige mice bearing human skin grafts are injected i.p. with human PBMC allogeneic to the donor skin, infiltrating T cells destroy human microvessels by day 21. Intradermal injection of human IL-11 (500 ng/day) delays the time course of graft microvessel loss without reducing the extent of T cell infiltration. Protective actions of IL-11 are most pronounced on day 15. IL-11 has no effect on T cell activation marker, effector molecule, cytokine expression, or endothelial ICAM-1 expression. IL-11 up-regulates the expression of survivin, a cytoprotective protein, in graft keratinocytes and endothelial cells. Topical application of survivin antisense oligonucleotide down-regulates survivin expression in both cell types and largely abrogates the protective effect of IL-11. We conclude that in this human transplant model, IL-11 exerts a cytoprotective rather than anti-inflammatory or immunomodulatory effect mediated through induction of survivin.
Independent Control ofLy49gAlleles: Implications for NK Cell Repertoire Selection and Tumor Cell Killing
Authors: Makrigiannis Andrew P. , Rousselle Etienne , Anderson Stephen K.
Published: April 2014

A novel murine NK cell-reactive mAb, AT8, was generated. AT8 recognizes Ly49G from 129/J, BALB/c, and related mouse strains, but does not bind to Ly49GB6. Costaining with AT8 and a Ly49GB6-restricted Ab (Cwy-3) provides the first direct evidence that Ly49G protein is expressed from both alleles on a significant proportion of NK cells from four different types of F1 hybrid mice. The observed level of biallelic Ly49G expression reproducibly followed the product rule in both freshly isolated and cultured NK cells. Surprisingly, the percentage of NK cells expressing both Ly49G alleles could be dramatically increased in vitro and in vivo through IL-2R- and IFN receptor-dependent signaling pathways, respectively. Unexpectedly, Ly49GB6+ NK cells in an H-2d, but not H-2b, background were more likely to lyse Dd+ and Chinese hamster ovary tumor cells than Ly49GBALB/129+ NK cells. Furthermore, Ly49GB6+ NK cells also proliferated to a higher degree in response to poly(I:C) than NK cells expressing a non-Ly49GB6 allele in an H-2d, but not H-2b, background. These results suggest that Ly49GB6 has a lower affinity for H-2Dd than Ly49GBALB/129, and the genetic background calibrates the responsiveness of NK cells bearing self-specific Ly49. Other H-2Dd receptors on the different Ly49G+ NK cell subsets were unequally coexpressed, possibly explaining the disparate responses of Ly49GB6+ NK cells in different hybrid mice. These data indicate that the stochastic mono- and biallelic expression of divergent Ly49G alleles increases the range of MHC affinities and the functional potential in the total NK cell population of heterozygous mice.
Estrogen Preferentially Promotes the Differentiation of CD11c+ CD11bintermediate Dendritic Cells from Bone Marrow Precursors
Authors: Paharkova-Vatchkova Vladislava , Maldonado Ruben , Kovats Susan
Published: April 2014

Sex biases in autoimmunity and infection suggest that steroid sex hormones directly modulate immune cells. We show in this study that 17-β-estradiol (E2) promotes the differentiation of functional dendritic cells (DC) from murine bone marrow precursor cells. Remarkably, ex vivo DC differentiation was inhibited in steroid hormone-deficient medium, and was restored by addition of physiological amounts of E2, but not dihydrotestosterone. DC differentiation was inhibited by the estrogen receptor (ER) antagonists ICI 182,780 and tamoxifen, and from ERα−/− bone marrow cells, indicating that E2 acted via ERs. E2 addition was most effective in promoting DC differentiation immediately ex vivo, but did not increase DC proliferation. E2 treatment specifically promoted differentiation of a CD11c+ CD11bint DC population that displayed high levels of cell surface MHC class II and CD86, suggesting that E2 could augment numbers of potent APC. DC that differentiated in E2-supplemented medium were fully functional in their capability to mediate presentation of self and foreign Ags and stimulate the proliferation of naive CD4+ T cells. The requirement for estrogen during DC differentiation suggests a mechanism by which E2 levels in peripheral tissues might modulate both the number and functional capabilities of DC in vivo, thereby influencing immune responses.
Production of Donor T Cells Is Critical for Induction of Donor-Specific Tolerance and Maintenance of Chimerism
Authors: Xu Hong , Chilton Paula M. , Huang Yiming , Schanie Carrie L. , Ildstad Suzanne T.
Published: April 2014

Nonmyeloablative conditioning has significantly reduced the morbidity associated with bone marrow transplantation. The donor hemopoietic cell lineage(s) responsible for the induction and maintenance of tolerance in nonmyeloablatively conditioned recipients is not defined. In the present studies we evaluated which hemopoietic stem cell-derived components are critical to the induction of tolerance in a total body irradiation-based model. Recipient B10 mice were pretreated with mAbs and transplanted with allogeneic B10.BR bone marrow after conditioning with 100–300 cGy total body irradiation. The proportion of recipients engrafting increased in a dose-dependent fashion. All chimeric recipients exhibited multilineage donor cell production. However, induction of tolerance correlated strictly with early production of donor T cells. The chimeras without donor T cells rejected donor skin grafts and demonstrated strong antidonor reactivity in vitro, while possessing high levels of donor chimerism. These animals lost chimerism within 8 mo. Differentiation into T cells was aborted at a prethymic stage in recipients that did not produce donor T cells. Moreover, donor Ag-driven clonal deletion of recipient T cells occurred only in chimeras with donor T cells. These results demonstrate that donor T cell production is critical in the induction of transplantation tolerance and the maintenance of durable chimerism. In addition, donor T cell production directly correlates with the deletion of potentially alloreactive cells.
ζ-Associated Protein of 70 kDa (ZAP-70), but Not Syk, Tyrosine Kinase Can Mediate Apoptosis of T Cells through the Fas/Fas Ligand, Caspase-8 and Caspase-3 Pathways
Authors: Zhong Lingwen , Wu Chun-Hua , Lee Wen-Hui , Liu Chih-Pin
Published: April 2014

The TCR ζ-chain-associated protein of 70 kDA (ZAP-70) and Syk tyrosine kinases play critical roles in regulating TCR-mediated signal transduction. They not only share some overlapped functions but also may play unique roles in regulating the function and development of T cells. However, it is not known whether they have different effects on the activation and activation-induced cell death of T cells. To address this question, we generated cDNAs encoding chimeric molecules that a tailless TCR ζ-chain was directly linked to truncated ZAP-70 (Z/ZAP) or Syk (Z/Syk) molecules lacking the two Src homology 2 domains. Transfection of these molecules into ζ-chain-deficient cells restored their TCR expression. In addition, Z/ZAP and Z/Syk transfectants but not control cells demonstrated kinase activities in phosphorylating an exogenous substrate specific for ZAP-70 and Syk kinases. Z/ZAP transfectants activated through TCRs underwent a faster time course of apoptosis and had a greater percentage of apoptotic cells than that of Z/Syk and control cells. Activated Z/ZAP transfectants increased Fas and Fas ligand (FasL) expression 3- and 40-fold, respectively. Blocking of the Fas/FasL interaction could inhibit the apoptosis of Z/ZAP transfectants. In contrast, although activated Z/Syk transfectants could increase FasL expression, their Fas expression actually decreased and the percentage of apoptotic cells did not increase. Further studies of the mechanisms revealed that activation of Z/ZAP but not Z/Syk transfectants resulted in rapid activation of caspase-3 and caspase-8 that could also be inhibited by blocking Fas/FasL interaction. These results demonstrated that ZAP-70 and Syk play distinct roles in T cell activation and activation-induced cell death.
Early Adenoviral Gene Expression Mediates Immunosuppression by Transduced Dendritic Cell (DC): Implications for Immunotherapy Using Genetically Modified DC
Authors: Tuettenberg Andrea , Jonuleit Helmut , Tüting Thomas , Brück Jürgen , Biermann Volker , Kochanek Stefan , Knop Jürgen , Enk Alexander H.
Published: April 2014

Long-lasting, high-level gene expression in the absence of a toxic or inflammatory response to viral Ags is necessary for the successful application of genetically modified dendritic cell (DC). We previously demonstrated that efficient transduction of mature DC using ΔE1ΔE3 adenoviruses suppressed their stimulatory capacity for T cells. The current study was designed to investigate in more detail the suppressive effect of Ad-DC. We demonstrate that immunosuppression is not mediated by alterations in the T cell phenotype or cytokine profiles released by stimulated T cells. Also DC phenotypes are not affected. However, we demonstrate a cell cycle arrest of the T cell population stimulated by adenovirally transduced DC. Surprisingly, only freshly transduced DC are perturbed in their stimulatory capacity. Experiments using cycloheximide to block early intracellular viral gene expression showed that viral genes expressed in DC are responsible for this transient immunosuppression. In agreement with these findings, high-capacity (gutless) Ad-vectors that differ in viral gene expression from conventional ΔE1ΔE3 adenovirus are suitable for an efficient transduction of human DC. DC transduced with gutless Ad-vectors showed a high allostimulatory capacity for CD4+ and CD8+ T cells. Thus, the immunosuppressive effect of ΔE1ΔE3 Ad-transduced mature DC seems to be the result of early viral gene expression in DC that can be prevented using gutless Ad-vectors for transduction. These results have important implications for the use of genetically modified DC for therapeutic application.