bluuebunny/crossref_metadata_2025 huggingface dataset:
CTLA-4 Is Not Required for Induction of CD8+ T Cell Anergy In Vivo
Authors: Frauwirth Kenneth A. , Alegre Maria-Luisa , Thompson Craig B.
Published: April 2014

Recent studies of T cell anergy induction have produced conflicting conclusions as to the role of the negative regulatory receptor, CTLA-4. Several in vivo models of tolerance have implicated the interaction of CTLA-4 and its ligands, B7.1 and B7.2, as an essential step in induction of anergy, while results from a number of other systems have indicated that signals from the TCR/CD3 complex alone are sufficient to induce T cell unresponsiveness. One explanation for this disparity is that the requirements for anergy induction depend closely on the details of the system: in vivo vs in vitro, route of stimulus administration, naive vs memory cells, CD4+ vs CD8+ cells, etc. To test this possibility, we established an in vivo anergy model using mice transgenic for the 2C TCR on a recombination-activating gene-2-deficient background, that either express or lack the CTLA-4 molecule. This system provides us with a very homogeneous pool of naive Ag-specific CD8+ T cells, allowing us to control some of the conditions mentioned above. We found that T cells from CTLA-4-deficient mice were anergized by injections of soluble antigenic peptide as efficiently as were CTLA-4-expressing cells. These results indicate that CTLA-4 is not universally required for in vivo T cell anergy induction and may point to distinctions between regulation of peripheral tolerance in CD4+ and CD8+ T cells.
Extracellular Nicotinamide Adenine Dinucleotide Induces T Cell Apoptosis In Vivo and In Vitro
Authors: Liu Zhang-Xu , Azhipa Olga , Okamoto Shigefumi , Govindarajan Sugantha , Dennert Gunther
Published: April 2014

Incubation of mouse T cells expressing the cell surface enzyme ADP ribosyltransferase with nicotinamide adenine dinucleotide (NAD) had been reported to cause ADP ribosylation of cell surface molecules, inhibition of transmembrane signaling, and suppression of immune responses. In this study, we analyze the reasons for these effects and report that contact of T cells with NAD causes cell death. Naive T cells when incubated with NAD and adoptively transferred into semiallogeneic mice fail to cause graft-vs-host disease, and when injected into syngeneic, T cell-deficient recipients do not reconstitute these mice. Rather, they accumulate in the liver, leading to an increase of apoptotic lymphocytes in this organ. Similar effects are induced by injection of NAD, shown to cause a dramatic increase of apoptotic CD3+, CD4+, and CD8+ cells in the liver. Consistent with this, in vitro incubation of naive T cells with NAD is shown to induce apoptosis. In contrast, no cell death is demonstrable when T cells are activated before incubation with NAD. It is concluded that ecto-NAD, as substrate of ADP ribosyltransferase, acts on naive, but not on activated CD69+ T cells.
Overexpression of AML1 Transcription Factor Drives Thymocytes into the CD8 Single-Positive Lineage
Authors: Hayashi Keitaro , Abe Natsumi , Watanabe Toshio , Obinata Masuo , Ito Mamoru , Sato Takehito , Habu Sonoko , Satake Masanobu
Published: April 2014

To understand the gene regulation involved in the development of single-positive (SP) thymocytes, we generated transgenic mice in which the AML1 transcription factor is overexpressed. In these mice the number of CD8 SP thymocytes was greatly increased, and this continued to be true even when MHC class I was absent. This promotion to the CD8 SP lineage was not, however, observed when both class I and class II were absent. Furthermore, even thymocytes carrying MHC class II-restricted TCR differentiated into the CD8 SP lineage when AML1 was overexpressed. The selected CD8 SP cells were, however, unable to mature, as judged by the expression level of heat-stable Ag. Thus, overexpression of AML1 is able to skew class II-restricted thymocytes into the CD8 SP lineage, but not to drive the maturation of resulting selected CD8 SP cells.
Duration and Strength of Extracellular Signal-Regulated Kinase Signals Are Altered During Positive Versus Negative Thymocyte Selection
Authors: Mariathasan Sanjeev , Zakarian Arsen , Bouchard Denis , Michie Alison M. , Zúñiga-Pflücker Juan Carlos , Ohashi Pamela S.
Published: April 2014

During thymocyte development, high-affinity/avidity TCR engagement leads to the induction of negative selection and apoptosis, while lower TCR affinity-avidity interactions lead to positive selection and survival. To elucidate how these extracellular interactions are translated into intracellular signals that distinguish between positive and negative selection, we developed a culture system in which naive double-positive thymocytes were either induced to differentiate along the CD8+ lineage pathway or were triggered for clonal deletion. Using this system, we show that sustained low level activation of extracellular signal-regulated kinases (ERKs) promotes positive selection, whereas strong but transient ERK activation is coupled with negatively selecting stimuli. Importantly, similar ERK activation profiles were demonstrated during positive selection for strong agonist ligands presented at low concentrations or weak agonist ligands. This is consistent with the affinity/avidity model and a role for strong or weak agonists during positive selection. Surprisingly, the addition of a pharmacological inhibitor which blocks ERK activation prevented the induction of negative selection. These data suggest that the duration and strength of the TCR signal is involved in discriminating between positive and negative selection.
1α,25-Dihydroxyvitamin D3 Has a Direct Effect on Naive CD4+ T Cells to Enhance the Development of Th2 Cells
Authors: Boonstra André , Barrat Franck J. , Crain Chad , Heath Victoria L. , Savelkoul Huub F. J. , O’Garra Anne
Published: April 2014

1α,25-Dihydroxyvitamin D3 (vitD3) is an immunoregulatory hormone with beneficial effects on Th1 mediated autoimmune diseases. Although the inhibitory effects of vitD3 on macrophages and dendritic cells are well documented, any direct effects of vitD3 on Th cell development are not clearly defined. Using CD4+Mel14+ T cells derived from mice on a BALB/c and a C57BL/6 genetic background we examined the effect of vitD3 on Th cell development. We demonstrated that vitD3 affects Th cell polarization by inhibiting Th1 (IFN-γ production) and augmenting Th2 cell development (IL-4, IL-5, and IL-10 production). These effects were observed in cultures driven with splenic APC and Ag, as well as with anti-CD3 and anti-CD28 alone, indicating that CD4+ cells can also be direct targets for vitD3. The enhanced Th2 development by vitD3 was found in both BALB/c and C57BL/6 mice. An increased expression of the Th2-specific transcription factors GATA-3 and c-maf correlated with the increased production of Th2 cytokines after vitD3 treatment. The vitD3-induced effects were largely mediated via IL-4, because neutralization of IL-4 almost completely abrogated the augmented Th2 cell development after vitD3 treatment. These findings suggest that vitD3 acts directly on Th cells and can, in the absence of APC, enhance the development of a Th2 phenotype and augment the expression of the transcription factors c-maf and GATA-3. Our findings suggest that the beneficial effects of vitD3 in autoimmune diseases and transplantation operate through prevention of strong Th1 responses via the action on the APC, while simultaneously directly acting on the T cell to enhance Th2 cell development.
Preferential Blockade of CD8+ T Cell Responses by Administration of Anti-CD137 Ligand Monoclonal Antibody Results in Differential Effect on Development of Murine Acute and Chronic Graft-Versus-Host Diseases
Authors: Nozawa Kazuhisa , Ohata Junko , Sakurai Jinkyo , Hashimoto Hiroshi , Miyajima Hiroaki , Yagita Hideo , Okumura Ko , Azuma Miyuki
Published: April 2014

We investigated the effect of CD137 costimulatory blockade in the development of murine acute and chronic graft-vs-host diseases (GVHD). The administration of anti-CD137 ligand (anti-CD137L) mAb at the time of GVHD induction ameliorated the lethality of acute GVHD, but enhanced IgE and anti-dsDNA IgG autoantibody production in chronic GVHD. The anti-CD137L mAb treatment efficiently inhibited donor CD8+ T cell expansion and IFN-γ expression by CD8+ T cells in both GVHD models and CD8+ T cell-mediated cytotoxicity against host-alloantigen in acute GVHD. However, a clear inhibition of donor CD4+ T cell expansion and activation has not been observed. On the contrary, in chronic GVHD, the number of CD4+ T cells producing IL-4 was enhanced by anti-CD137L mAb treatment. This suggests that the reduction of CD8+ T cells producing IFN-γ promotes Th2 cell differentiation and may result in exacerbation of chronic GVHD. Our results highlight the effective inactivation of CD8+ T cells and the lesser effect on CD4+ T cell inactivation by CD137 blockade. Intervention of the CD137 costimulatory pathway may be beneficial for some selected diseases in which CD8+ T cells are major effector or pathogenic cells. Otherwise, a combinatorial approach will be required for intervention of CD4+ T cell function.
Clonally Diverse CTL Response to a Dominant Viral Epitope Recognizes Potential Epitope Variants
Authors: Charini William A. , Kuroda Marcelo J. , Schmitz Jörn E. , Beaudry Kristin R. , Lin Wenyu , Lifton Michelle A. , Krivulka Georgia R. , Necker Antje , Letvin Norman L.
Published: April 2014

RNA viruses undergo rapid sequence variation as the result of error-prone RNA replication mechanisms. When viable mutations arise in RNA regions encoding B or T cell epitopes, mutant viruses that can evade immune detection may be selected. In the carefully studied CTL response to the Gag p11C(C-M) epitope in SIVmac-infected Mamu-A*01+ rhesus monkeys, it has been shown that CTL recognition of that epitope can occur even in the face of accruing mutations. To explore the underlying mechanism for this breadth of recognition, we have constructed Mamu-A*01 tetramers which discriminate T cells specific for epitope variants. Using these reagents we have defined discrete subsets of p11C(C-M)-specific T cells that cross-react with cells presenting variant peptides. We have found that individual Mamu-A*01+ monkeys differ functionally in their ability to recognize epitope variants despite consistently strong recognition of the p11C(C-M) epitope. This functional difference is accounted for by the relative number of variant-specific T cells and by differences in the functionally relevant TCR repertoire of the infected monkeys. We have also found that monkeys immunized with DNA vaccine constructs encoding only the wild-type epitope sequence develop p11C(C-M)-specific CTL cross-reactive with variant peptides. Thus, cross-reactive CTL do not merely arise secondary to the emergence and immune presentation of viral CTL escape mutants but rather arise de novo following priming with a dominant epitope peptide sequence. Taken together, our results support the concept that the CTL response to a dominant viral epitope, although highly focused, can be clonally diverse and recognize potential epitope variants.
Embryonic Thymic Epithelium Naturally Devoid of APCs Is Acutely Rejected in the Absence of Indirect Recognition
Authors: Pimenta-Araujo Ricardo , Mascarell Laurent , Huesca Michèle , Cumano Ana , Bandeira Antonio
Published: April 2014

Transplants of tissues depleted of passenger leukocytes are upon in vitro culture usually accepted in allogeneic recipients. Accordingly, fully allogeneic embryonic thymic epithelium was suggested to be poorly immunogenic. However, this tissue is capable of inducing donor-specific tolerance to peripheral tissues, when restoring T cell development in nude mice, through the production of regulatory cells. In the present work, adult immunocompetent allogeneic recipients were grafted with embryonic tissues isolated at stages before hemopoietic colonization or even before the establishment of circulation. Allogeneic thymic epithelium of day 10 embryos and heart primordium of day 8 embryonic donors were always rejected. Acute rejection of the thymic anlagen takes place in less than 12 days, with maximal CD4+ and CD8+ T cell infiltrates at 10 days post-transplant. In addition, a significant infiltrate of NK1.1+ cells is observed, although without any essential role in this process. Furthermore, recipients lacking the indirect pathway of Ag presentation to CD4+ T cells do not reveal any significant delay in rejection, even when CD8+ T cells are also eliminated. Thus, our experimental approach reveals acute allograft rejection in the absence of all known pathways of naive T cell activation and therefore unveils a novel graft rejection mechanism that should be mediated by direct recognition of parenchymal cells. Given the importance of dendritic cells in naive T cell activation, it is likely that cross-reactive memory T cells may also drive rejection.
Caspase 8 Activity in Membrane Blebs After Anti-Fas Ligation
Authors: Packard Beverly Z. , Komoriya Akira , Brotz Tilmann M. , Henkart Pierre A.
Published: April 2014

Previous studies of thymocyte apoptosis using a series of cell-permeable fluorogenic peptide substrates showed that Fas cross-linking triggered a caspase cascade in which cleavage of the IETDase (caspase 8-selective) substrate was the earliest caspase activity measured by flow cytometry. This result was expected in light of the abundant evidence for caspase 8 activation as an initiating event in the Fas death pathway. However, when apoptosis was induced by anti-Fas in CTL and the caspase cascade examined by this approach, IETDase activation followed increases in LEHDase, YVHDase, and VEIDase activities (selective for caspases 9, 1, and 6, respectively). When examined by confocal microscopy, anti-Fas-treated CTL showed the early appearance of IETDase-containing plasma membrane vesicles and their release from the CTL surface, followed by activation of other caspase activities in the cell interior. Since these vesicles were not included in the flow cytometry analysis, the early IETDase activity had been underestimated. In contrast to anti-Fas, induction of apoptosis in these CTL by IL-2 withdrawal resulted in early IETDase activity in the cytoplasm, with no plasma membrane vesiculation. Thus, anti-Fas-induced initiation of caspase activity at the plasma membrane may in some cells result in local proteolysis of submembrane proteins, leading to generation of membrane vesicles that are highly enriched in active caspase 8.
Lipopolysaccharides from Distinct Pathogens Induce Different Classes of Immune Responses In Vivo
Authors: Pulendran Bali , Kumar Padmasini , Cutler Christopher W. , Mohamadzadeh Mansour , Van Dyke Thomas , Banchereau Jacques
Published: April 2014

The adaptive immune system has evolved distinct responses against different pathogens, but the mechanism(s) by which a particular response is initiated is poorly understood. In this study, we investigated the type of Ag-specific CD4+ Th and CD8+ T cell responses elicited in vivo, in response to soluble OVA, coinjected with LPS from two different pathogens. We used Escherichia coli LPS, which signals through Toll-like receptor 4 (TLR4) and LPS from the oral pathogen Porphyromonas gingivalis, which does not appear to require TLR4 for signaling. Coinjections of E. coli LPS + OVA or P. gingivalis LPS + OVA induced similar clonal expansions of OVA-specific CD4+ and CD8+ T cells, but strikingly different cytokine profiles. E. coli LPS induced a Th1-like response with abundant IFN-γ, but little or no IL-4, IL-13, and IL-5. In contrast, P. gingivalis LPS induced Th and T cell responses characterized by significant levels of IL-13, IL-5, and IL-10, but lower levels of IFN-γ. Consistent with these results, E. coli LPS induced IL-12(p70) in the CD8α+ dendritic cell (DC) subset, while P. gingivalis LPS did not. Both LPS, however, activated the two DC subsets to up-regulate costimulatory molecules and produce IL-6 and TNF-α. Interestingly, these LPS appeared to have differences in their ability to signal through TLR4; proliferation of splenocytes and cytokine secretion by splenocytes or DCs from TLR4-deficient C3H/HeJ mice were greatly impaired in response to E. coli LPS, but not P. gingivalis LPS. Therefore, LPS from different bacteria activate DC subsets to produce different cytokines, and induce distinct types of adaptive immunity in vivo.
Identification of Tumor-Infiltrating Macrophages as the Killers of Tumor Cells After Immunization in a Rat Model System
Authors: Bonnotte Bernard , Larmonier Nicolas , Favre Nathalie , Fromentin Annie , Moutet Monique , Martin Monique , Gurbuxani Sandeep , Solary Eric , Chauffert Bruno , Martin François
Published: April 2014

Immunization can prevent tumor growth, but the effector cells directly responsible for tumor cell killing in immunized hosts remain undetermined. The present study compares tumor grafts that progress in naive syngeneic rats with the same grafts that completely regress in hosts preimmunized with an immunogenic cell variant. The progressive tumors contain only a few macrophages that remain at the periphery of the tumor without direct contact with the cancer cells. These macrophages do not kill tumor cells in vitro. In contrast, tumors grafted in immunized hosts and examined at the beginning of tumor regression show a dramatic infiltration with mature macrophages, many of them in direct contact with the cancer cells. These macrophages are strongly cytotoxic for the tumor cells in vitro. In contrast to macrophages, tumor-associated lymphocytes are not directly cytotoxic to the tumor cells, even when obtained from tumor-immune rats. However, CD4+ and CD8+ T cells prepared from the regressing tumors induce tumoricidal activity in splenic macrophages from normal or tumor-bearing rats and in macrophages that infiltrate progressive tumors. These results strongly suggest that the main tumoricidal effector cells in preimmunized rats are macrophages that have been activated by adjacent tumor-immune lymphocytes.
Differential Developmental Regulation and Functional Effects on Pre-TCR Surface Expression of Human pTαa and pTαb Spliced Isoforms
Authors: Ramiro Almudena R. , Navarro María N. , Carreira Aura , Carrasco Yolanda R. , de Yébenes Virginia G. , Carrillo Graciela , San Millán José L. , Rubin Bent , Toribio María L.
Published: April 2014

Functional rearrangement at the TCRβ locus leads to surface expression on developing pre-T cells of a pre-TCR complex composed of the TCRβ-chain paired with the invariant pre-TCRα (pTα) chain and associated with CD3 components. Pre-TCR signaling triggers the expansion and further differentiation of pre-T cells into TCRαβ mature T cells, a process known as β selection. Besides the conventional pTα transcript (termed pTαa), a second, alternative spliced, isoform of the pTα gene (pTαb) has been described, whose developmental relevance remains unknown. In this study, phenotypic, biochemical, and functional evidence is provided that only pTαa is capable of inducing surface expression of a CD3-associated pre-TCR complex, which seems spontaneously recruited into lipid rafts, while pTαb pairs with and retains TCRβ intracellularly. In addition, by using real-time quantitative RT-PCR approaches, we show that expression of pTαa and pTαb mRNA spliced products is differentially regulated along human intrathymic development, so that pTαb transcriptional onset is developmentally delayed, but β selection results in simultaneous shutdown of both isoforms, with a relative increase of pTαb transcripts in β-selected vs nonselected pre-T cells in vivo. Relative increase of pTαb is also shown to occur upon pre-T cell activation in vitro. Taken together, our data illustrate that transcriptional regulation of pTα limits developmental expression of human pre-TCR to intrathymic stages surrounding β selection, and are compatible with a role for pTαb in forming an intracellular TCRβ-pTαb complex that may be responsible for limiting surface expression of a pTαa-containing pre-TCR and/or may be competent to signal from a subcellular compartment.
Genomic Characterization of LIGHT Reveals Linkage to an Immune Response Locus on Chromosome 19p13.3 and Distinct Isoforms Generated by Alternate Splicing or Proteolysis
Authors: Granger Steve W. , Butrovich Kris D. , Houshmand Pantea , Edwards Wilson R. , Ware Carl F.
Published: April 2014

LIGHT is a member of the TNF cytokine superfamily that signals through the lymphotoxin (LT)β receptor and the herpesvirus entry mediator. LIGHT may function as a costimulatory factor for the activation of lymphoid cells and as a deterrent to infection by herpesvirus, which may provide significant selective pressure shaping the evolution of LIGHT. Here, we define the molecular genetics of the human LIGHT locus, revealing its close linkage to the TNF superfamily members CD27 ligand and 4-1BB ligand, and the third complement protein (C3), which positions LIGHT within the MHC paralog on chromosome 19p13.3. An alternately spliced isoform of LIGHT mRNA that encodes a transmembrane-deleted form is detected in activated T cells and gives rise to a nonglycosylated protein that resides in the cytosol. Furthermore, membrane LIGHT is shed from the cell surface of human 293 T cells. These studies reveal new mechanisms involved in regulating the physical forms and cellular compartmentalization of LIGHT that may contribute to the regulation and biological function of this cytokine.
Structural Analysis of Mutants of High-Affinity and Low-Affinityp-Azophenylarsonate-Specific Antibodies Generated by Alanine Scanning of Heavy Chain Complementarity-Determining Region 2
Authors: Parhami-Seren Behnaz , Viswanathan Malini , Strong Roland K. , Margolies Michael N.
Published: April 2014

Alanine scanning was used to determine the affinity contributions of 10 side chain amino acids (residues at position 50–60 inclusive) of H chain complementarity-determining region 2 (HCDR2) of the somatically mutated high-affinity anti-p-azophenylarsonate Ab, 36–71. Each mutated H chain gene was expressed in the context of mutated (36–71L) and the unmutated (36–65L) L chains to also assess the contribution of L chain mutations to affinity. Combined data from fluorescence quenching, direct binding, inhibition, and capture assays indicated that mutating H:Tyr50 and H:Tyr57 to Ala in the 36–71 H chain results in significant loss of binding with both mutated (36–71L) or unmutated (36–65L) L chain, although the decrease was more pronounced when unmutated L chain was used. All other HCDR2 mutations in 36–71 had minimal effect on Ab affinity when expressed with 36–71 L chain. However, in the context of unmutated L chain, of H:Gly54 to Ala resulted in significant loss of binding, while Abs containing Asn52 to Ala, Pro53 to Ala, or Ile58 to Ala mutation exhibited 4.3- to 7.1-fold reduced affinities. When alanine scanning was performed instead on certain HCDR2 residues of the germline-encoded (unmutated) 36–65 Ab and expressed with unmutated L chain as Fab in bacteria, these mutants exhibited affinities similar to or slightly higher than the wild-type 36–65. These findings indicate an important role of certain HCDR2 side chain residues on Ab affinity and the constraints imposed by L chain mutations in maintaining Ag binding.
Loss of PU.1 Expression Following Inhibition of Histone Deacetylases
Authors: Laribee R. Nicholas , Klemsz Michael J.
Published: April 2014

Altering chromatin structure by blocking histone deacetylase activity with specific inhibitors such as trichostatin A can result in an up-regulation of gene expression. In this report, however, we show that expression of the ETS domain transcription factor PU.1 is down-regulated in cells following the addition of trichostatin A. The loss of PU.1 is seen at both the mRNA and protein levels in multiple cell lines and is reversible following removal of the drug. More importantly, we show that the loss of PU.1 results in a loss of PU.1 target gene expression, including CD11b, c-fms, Toll-like receptor 4, and scavenger receptor. Chromatin immunoprecipitation analysis of cells treated with trichostatin A showed a significant increase in the acetylation of histone H4, but not histone H3, across approximately 650 bp of the PU.1 promoter region. Our data suggest that the consequences of using drugs that inhibit histone deacetylase activity may be a loss of blood cell development and/or function due to a block in PU.1 gene expression.
The MHC-Specific Enhanceosome and Its Role in MHC Class I and β2-Microglobulin Gene Transactivation
Authors: Gobin Sam J. P. , van Zutphen Marlijn , Westerheide Sandy D. , Boss Jeremy M. , van den Elsen Peter J.
Published: April 2014

The promoter regions of MHC class I and β2-microglobulin (β2m) genes posses a regulatory module consisting of S, X, and Y boxes, which is shared by MHC class II and its accessory genes. In this study we show that, similar to MHC class II, the SXY module in MHC class I and β2m promoters is cooperatively bound by a multiprotein complex containing regulatory factor X, CREB/activating transcription factor, and nuclear factor Y. Together with the coactivator class II transactivator this multiprotein complex drives transactivation of these genes. In contrast to MHC class II, the multiprotein complex has an additional function in the constitutive transactivation of MHC class I and β2m genes. The requirement for all transcription factors in the complex and correct spacing of the binding sites within the SXY regulatory module for complex formation and functioning of this multiprotein complex strongly suggests that this complex can be regarded as a bona fide enhanceosome. The general coactivators CREB binding protein, p300, general control nonderepressible-5, and p300/CREB binding protein-associated factor exert an ancillary function in MHC class I and β2m transactivation, but exclusively through the class II transactivator component of this enhanceosome. Thus, the SXY module is the basis for a specific enhanceosome important for the constitutive and inducible transactivation of MHC class I and β2m genes.
The Vesicular Stomatitis Virus Matrix Protein Inhibits Glycoprotein 130-Dependent STAT Activation
Authors: Terstegen Lara , Gatsios Petros , Ludwig Stephan , Pleschka Stephan , Jahnen-Dechent Willi , Heinrich Peter C. , Graeve Lutz
Published: April 2014

Infection of cells by vesicular stomatitis virus (VSV) results in the inhibition of host transcription. We show in this study that infection of HeLa cells with VSV leads to a strongly diminished activation of STAT3 and STAT1 by the inflammatory cytokine IL-6. This effect was mimicked by forced expression of a single viral protein, the matrix (M)-protein of VSV, which blocked STAT activation via chimeric receptors containing the cytoplasmic domain of the IL-6 signal transducer gp130. Western blot analysis revealed that VSV M-protein did not inhibit the nuclear translocation of activated STAT3 but did inhibit its tyrosine phosphorylation. Inhibition of STAT activation was not dependent on tyrosine 759 of the IL-6 signal transducer gp130, suggesting that the inhibitory action of VSV M-protein is not mediated by the induction of the suppressor of cytokine signaling 3. VSV M-protein inhibited gene transcription from cotransfected α2-macroglobulin or antichymotrypsin promoter/luciferase reporter constructs which contain STAT3-binding sites. However, transcription from a STAT5-dependent construct was not negatively affected. In conclusion, our data suggest that infection by VSV and specifically overexpression of the viral M-protein interferes with an important signaling pathway necessary for triggering antiviral and inflammatory responses.
Sandfly Maxadilan Exacerbates Infection with Leishmania major and Vaccinating Against It Protects Against L. major Infection
Authors: Morris Robin V. , Shoemaker Charles B. , David John R. , Lanzaro Gregory C. , Titus Richard G.
Published: April 2014

Bloodfeeding arthropods transmit many of the world’s most serious infectious diseases. Leishmania are transmitted to their mammalian hosts when an infected sandfly probes in the skin for a bloodmeal and injects the parasite mixed with its saliva. Arthropod saliva contains molecules that affect blood flow and modulate the immune response of the host. Indeed, sandfly saliva markedly enhances the infectivity of L. major for its host. If the salivary molecule(s) responsible for this phenomenon was identified, it might be possible to vaccinate the host against this molecule and thereby protect the host against infection with Leishmania. Such an approach represents a novel means of controlling arthropod-borne disease transmission. Here, we report that a single molecule, maxadilan, in sandfly saliva can exacerbate infection with L. major to the same degree as whole saliva, and that vaccinating against maxadilan protects mice against infection with L. major.
Escherichia coliBraun Lipoprotein Induces a Lipopolysaccharide-Like Endotoxic Response from Primary Human Endothelial Cells
Authors: Neilsen Paul O. , Zimmerman Guy A. , McIntyre Thomas M.
Published: April 2014

All bacteria contain proteins in which their amino-terminal cysteine residue is modified with N-acyl S-diacylglycerol functions, and peptides and proteins bearing this modification are immunomodulatory. The major outer membrane lipoprotein of Escherichia coli, the Braun lipoprotein (BLP), is the prototypical triacylated cysteinyl-modified protein. We find it is as active as LPS in stimulating human endothelial cells to an inflammatory phenotype, and a BLP-negative mutant of E. coli was less inflammatory than its parental strain. While the lipid modification was essential, the lipidated protein was more potent than a lipid-modified peptide. BLP associates with CD14, but this interaction, unlike that with LPS, was not required to elicit endothelial cell activation. BLP stimulated endothelial cell E-selectin surface expression, IL-6 secretion, and up-regulation of the same battery of cytokine mRNAs induced by LPS. Quantitative microarray analysis of 4400 genes showed the same 30 genes were induced by BLP and LPS, and that there was near complete concordance in the level of gene induction. We conclude that the lipid modification of at least one abundant Gram-negative protein is essential for endotoxic activity, but that the protein component also influences activity. The equivalent potency of BLP and LPS, and their complete concordance in the nature and extent of endothelial cell activation show that E. coli endotoxic activity is not due to just LPS. The major outer membrane protein of E. coli is a fully active endotoxic agonist for endothelial cells.
Immunization of Mice Against West Nile Virus with Recombinant Envelope Protein
Authors: Wang Tian , Anderson John F. , Magnarelli Louis A. , Wong Susan J. , Koski Raymond A. , Fikrig Erol
Published: April 2014

West Nile (WN) virus is a mosquito-borne flavivirus that emerged in the United States in 1999 and can cause fatal encephalitis. Envelope (E) protein cDNA from a WN virus isolate recovered from Culex pipiens in Connecticut was expressed in Escherichia coli. The recombinant E protein was purified and used as Ag in immunoblot assays and immunization experiments. Patients with WN virus infection had Abs that recognized the recombinant E protein. C3H/HeN mice immunized with E protein developed E protein Abs and were protected from infection with WN virus. Passive administration of E protein antisera was also sufficient to afford immunity. E protein is a candidate vaccine to prevent WN virus infection.
The Schistosome Oligosaccharide Lacto-N-neotetraose Expands Gr1+ Cells That Secrete Anti-inflammatory Cytokines and Inhibit Proliferation of Naive CD4+ Cells: A Potential Mechanism for Immune Polarization in Helminth Infections
Authors: Terrazas Luis I. , Walsh Kristen L. , Piskorska Danuta , McGuire Edward , Harn Donald A.
Published: April 2014

Immunomodulatory oligosaccharides found on helminths also are found in human milk, and both helminths and milk have been shown to be immunosuppressive. We have been examining the immunomodulatory capabilities of two oligosaccharides expressed in milk and on helminth parasites, lacto-N-fucopentaose III and lacto-N-neotetraose (LNnT). In an attempt to dissect mechanisms that lead to Th2 polarization and immune suppression, we examined the early response in mice to the glycoconjugate LNnT-Dextran (LNnT-Dex). We found that injection of LNnT-Dex expanded a cell population, phenotypically defined as Gr1+/CD11b+/F4/80+, as early as 2 h after injection. Examination of spontaneous cytokine production showed that this Gr1+/F4/80+ population of cells spontaneously produced low levels of proinflammatory cytokines, but higher levels of IL-10 and TGF-β ex vivo, compared to peritoneal cells from mice injected with Dex. Gr1+ cells adoptively suppressed naive CD4+ T cell proliferation in vitro in response to anti-CD3/CD28 Ab stimulation. Suppression of naive CD4+ cells involved cell contact and was dependent on IFN-γ and NO, with a discrete role played by IL-10. Coculture of naive CD4+T cells with Gr1+ suppressor cells did not lead to CD4+ T cell apoptosis, although it did imprint on naive CD4+ T cells a response characterized by lower levels of IFN-γ, coincident with increased IL-13 production. Our results suggest that both human milk and helminth parasites may share a ligand-specific mechanism involved in the generation of anti-inflammatory mediators that suppress Th1-type and inflammatory responses.
A Proinflammatory Role of IL-18 in the Development of Spontaneous Autoimmune Disease
Authors: Esfandiari Ehsanollah , McInnes Iain B. , Lindop George , Huang Fang-Ping , Field Max , Komai-Koma Mousa , Wei Xiao-qing , Liew Foo Y.
Published: April 2014

Serum from patients with systemic lupus erythematosus (SLE) contained significantly higher concentrations of IL-18 than normal individuals. MRL/lpr mice, which develop spontaneous lupus-like autoimmune disease, also had higher serum levels of IL-18 than wild-type MRL/++ mice. Daily injections of IL-18 or IL-18 plus IL-12 resulted in accelerated proteinuria, glomerulonephritis, vasculitis, and raised levels of proinflammatory cytokines in MRL/lpr mice. IL-18-treated MRL/lpr mice also developed a “butterfly” facial rash resembling clinical SLE. In contrast, MRL/lpr mice treated with IL-18 plus IL-12 did not develop a facial rash. The facial lesion in the IL-18-treated mice showed epidermal thickening with intense chronic inflammation accompanied by increased apoptosis, Ig deposition, and early systemic Th2 response compared with control or IL-12 plus IL-18-treated mice. These data therefore show that IL-18 is an important mediator of lupus-like disease and may thus be a novel target for therapeutic intervention of spontaneous autoimmune diseases.
Novel Small Molecule Inhibitor of C1s Exerts Cardioprotective Effects in Ischemia-Reperfusion Injury in Rabbits
Authors: Buerke Michael , Schwertz Hansjörg , Seitz Werner , Meyer Jürgen , Darius Harald
Published: April 2014

Myocardial ischemia-reperfusion injury can be related to complement activation with generation of chemotactic agents, adhesion molecule expression, release of cytokines and oxygen-derived free radicals, and subsequent neutrophil accumulation. In the present study the cardioprotective effects of a novel highly selective small molecule C1s inhibitor (C1s-INH-248, Knoll) were examined in a rabbit model of myocardial ischemia (I) and reperfusion (R; i.e., 60 min I + 180 min R). In in vitro tests (enzyme activity and SRBC lysis) C1s-INH-248 demonstrated profound inhibitory potency. In vivo C1s-INH-248 (1 mg/kg body weight) administered 5 min before reperfusion significantly attenuated myocardial injury (31.9 ± 2.5 vs 8.9 ± 1.6% necrosis/area at risk; p < 0.01). The cardioprotective effect was dose dependent. The reduction of myocardial injury was also observed as diminished plasma creatine kinase activity in C1s-INH-248-treated animals (70.7 ± 6.8 vs 45.1 ± 3.9 U/g protein after 3 h of reperfusion, p < 0.05). Further, cardiac myeloperoxidase activity (i.e., a marker of PMN accumulation) in the ischemic and necrotic area was significantly reduced following C1s-INH-248 treatment (1.31 ± 0.23 vs 0.4 ± 0.05 U/100 mg tissue in necrotic area, p < 0.01). Thus, blocking the classical complement pathway with a highly specific and potent synthetic inhibitor of the activated C1 complex appears to be an effective mean to preserve ischemic myocardium from injury following reperfusion.
Resistance to Fas-Mediated Apoptosis in EBV-Infected B Cell Lymphomas Is Due to Defects in the Proximal Fas Signaling Pathway
Authors: Snow Andrew L. , Chen Linda J. , Nepomuceno Ronald R. , Krams Sheri M. , Esquivel Carlos O. , Martinez Olivia M.
Published: April 2014

Post-transplant lymphoproliferative disorder is characterized by the outgrowth of EBV-infected B cell lymphomas in immunosuppressed transplant recipients. Using a panel of EBV-infected spontaneous lymphoblastoid cell lines (SLCL) derived from post-transplant lymphoproliferative disorder patients, we assessed the sensitivity of such lymphomas to Fas-mediated cell death. Treatment with either an agonist anti-Fas mAb or Fas ligand-expressing cells identifies two subsets of SLCL based on their sensitivity or resistance to Fas-driven apoptosis. Fas resistance in these cells cannot be attributed to reduced Fas expression or to mutations in the Fas molecule itself. In addition, all SLCL are sensitive to staurosporine-induced cell death, indicating that there is no global defect in apoptosis. Although all SLCL express comparable levels of Fas signaling molecules including Fas-associated death domain protein, caspase 8, and caspase 3, Fas-resistant SLCL exhibit a block in Fas-signaling before caspase 3 activation. In two SLCL, this block results in impaired assembly of the death-inducing signaling complex, resulting in reduced caspase 8 activation. In a third Fas-resistant SLCL, caspase 3 activation is hindered despite intact death-inducing signaling complex formation and caspase 8 activation. Whereas multiple mechanisms exist by which tumor cells can evade Fas-mediated apoptosis, these studies suggest that the proximal Fas-signaling pathway is impeded in Fas-resistant post-transplant lymphoproliferative disorder-associated EBV+ B cell lymphomas.
Diversity of the Human Allergen-Specific T Cell Repertoire Associated with Distinct Skin Test Reactions: Delayed-Type Hypersensitivity-Associated Major Epitopes Induce Th1- and Th2-Dominated Responses
Authors: Woodfolk Judith A. , Platts-Mills Thomas A. E.
Published: April 2014

Distinct immune responses in humans to the Trichophyton rubrum Ag, Tri r 2, are associated with different patterns of T cell epitope recognition based on in vitro proliferation to peptides derived from this 29-kDa protein. Specifically, the amino-terminal immunodominant epitope, peptide 5 (P5), stimulates strong T cell proliferative responses in subjects with delayed (DTH), but not immediate (IH) hypersensitivity skin tests. Evidence of a role for cytokines or changes in epitope recognition over time was examined in responses to Trichophyton using primary PBMC cultures established from seven IH and seven DTH subjects. Responses stimulated by Tri r 2 were dominated by the Th1 cytokine IFN-γ (IFN-γ:IL-5 ≥ 4:1) in five DTH subjects, even in the presence of Th2-dominated responses (IFN-γ:IL-5 ≤ 3:1) to a subset of major epitopes. Paradoxically, P5 induced IL-5 and IL-10 production in DTH, but not IH subjects (p = 0.003 (IL-5), p = 0.024 (IL-10)), with no significant difference in IFN-γ levels between the two groups. In cultures from IH responders, no IL-5 was measurable after stimulation with P6 and P7 (as well as P5); this region of the molecule was shown previously to stimulate markedly reduced T cell proliferation in these individuals. Repeat proliferation assays confirmed no change in the pattern of peptide recognition after ≥20 mo in IH or DTH subjects. We conclude that T cell repertoires associated with distinct immune responses to Tri r 2 can be distinguished based on Th2 cytokine induction by DTH-associated major epitopes localizing to the amino-terminal region of the molecule.
Transplacental Genetic Immunization After Intravenous Delivery of Plasmid DNA to Pregnant Mice
Authors: Okuda Kenji , Xin Ke-Qin , Haruki Atsushi , Kawamoto Susumu , Kojima Yoshitsugu , Hirahara Fumiki , Okada Hidechika , Klinman Dennis , Hamajima Kenji
Published: April 2014

A number of factors influence the development of tolerance, including the nature, concentration, and mode of Ag presentation to the immune system, as well as the age of the host. The studies were conducted to determine whether immunizing pregnant mice with liposome-encapsulated DNA vaccines had an effect on the immune status of their offspring. Two different plasmids (encoding Ags from HIV-1 and influenza virus) were administered i.v. to pregnant mice. We examined the uptake of plasmid DNA by the fetuses until the 21st postcoital day, but little such transfer occurred in early pregnancy. At 9.5 days postconception with cationic liposomes, injected plasmid was present in the tissues of the fetus, consistent with transplacental transfer. When the offspring of vaccinated dams were immunized with DNA vaccine, they mounted stronger Ag-specific immune responses than controls, and were protected against challenge by homologous influenza virus after vaccination. Moreover, such immune responses were strong in the offspring of mothers injected with DNA plasmid 9.5 days after coitus. These results suggest that DNA-vaccinated mothers confer the Ag-specific immunity to their progeny.
Induction of Type 1 Immune Pathology in the Brain Following Immunization Without Central Nervous System Autoantigen in Transgenic Mice With Astrocyte-Targeted Expression of IL-12
Authors: Lassmann Silke , Kincaid Carrie , Asensio Valérie C. , Campbell Iain L.
Published: April 2014

IL-12, a cytokine produced by microglia, may regulate cellular immunity at a localized level in the CNS. To investigate this further, we examined the consequences of peripheral immune stimulation without specific autoantigen in wild-type or transgenic (termed GF-IL12) mice with astrocyte production of the bioactive IL-12 p75 heterodimer. Active immunization with CFA and pertussis toxin, a procedure known to stimulate a robust type 1-biased immune response, produced CNS immune pathology from which GF-IL12 but not wild-type mice developed signs of clinical disease consisting of loss of activity, piloerection, mild tremor, and motor change. All immunized mice had some degree of mononuclear cell infiltration into the brain; however, the severity of this was markedly increased in GF-IL12 mice where leukocytes accumulated in perivascular and parenchymal locations. Accumulating cells consisted of CD4+ and CD8+ T cells and macrophage/microglia. Moreover, expression of cytokines (IFN-γ and TNF), chemokines (IFN-inducible protein-10 and RANTES), the immune accessory molecules, MHC class II, B7.2, ICAM-1 and VCAM-1, and NO synthase-2 was induced in the CNS of the GF-IL12 mice. Therefore, peripheral immunization of GF-IL12 but not wild-type mice can provoke active type 1 immunity in the brain—a process that does not require CNS-specific immunizing autoantigen. These findings indicate that the cytokine milieu of a tissue can dramatically influence the development of intrinsic immune responses and associated pathology.
Noncovalent Association with Stress Protein Facilitates Cross-Priming of CD8+ T Cells to Tumor Cell Antigens by Dendritic Cells
Authors: Kammerer Robert , Stober Detlef , Riedl Petra , Oehninger Claude , Schirmbeck Reinhold , Reimann Jörg
Published: April 2014

A viral oncogene carrying well-defined Kb/Db-restricted epitopes was expressed in a heat shock protein (hsp)-associated or nonassociated form in the murine tumor cells P815 and Meth-A. Wild-type SV40 large T-Ag (wtT-Ag) is expressed without stable hsp association; mutant (cytoplasmic cT-Ag) or chimeric (cT272-green fluorescent fusion protein) T-Ag is expressed in stable association with the constitutively expressed, cytosolic hsp73 (hsc70) protein. In vitro, remnants from apoptotic wtT-Ag- or cT-Ag-expressing tumor cells are taken up and processed by immature dendritic cells (DC), and the Kb/Db-binding epitopes T1, T2/3, and T4 of the T-Ag are cross-presented to CTL in a TAP-independent way. DC pulsed with remnants of transfected, apoptotic tumor cells cross-presented the three T-Ag epitopes more efficiently when they processed ATP-sensitive hsp73/cT-Ag complexes than when they processed hsp-nonassociated (native) T-Ag. In vivo, more IFN-γ-producing CD8+ T cells were elicited by a DNA vaccine that encoded hsp73-binding mutant T-Ag than by a DNA vaccine that encoded native, non-hsp-binding T-Ag. Three- to 5-fold higher numbers of T-Ag (T1-, T2/3-, or T4-) specific, Db/Kb-restricted IFN-γ-producing CD8+ T cells were primed during the growth of transfected H-2d Meth-A/cT tumors than during the growth of transfected Meth-A/T tumors in F1(b × d) hosts. Hence, the association of an oncogene with constitutively expressed, cytosolic hsp73 facilitates cross-priming in vitro and in vivo of CTL by DC that process material from apoptotic cells.
Ly-6A.2 Expression Regulates Antigen-Specific CD4+ T Cell Proliferation and Cytokine Production
Authors: Henderson S. Christine , Kamdar Mihir M. , Bamezai Anil
Published: April 2014

Ly-6 proteins appear to serve cell adhesion and cell signaling function, but the precise role of Ly-6A.2 in CD4+ T lymphocytes is still unclear. Overexpression of Ly-6A.2 in T lymphocytes has allowed us to analyze the influence of elevated Ly-6A.2 expression on T cell function. In this study we report reduced proliferation of CD4+ T cells overexpressing Ly-6A.2 in response to a peptide Ag. Moreover, the Ly-6A.2-overexpressing CD4+ cells generated elevated levels of IL-4, a key factor that propels the differentiation of naive CD4+ T cells into Th2 subset. The hyporesponsiveness of Ly-6A.2 transgenic CD4+ T cells is dependent on the interaction of Ly-6A.2 T cells with the APCs and can be reversed by blocking the interaction between Ly-6A.2 and a recently reported candidate ligand. Overexpression of Ly-6A.2 in CD4+ T cells reduced their Ca2+ responses to TCR stimulation, therefore suggesting effects of Ly-6A.2 signaling on membrane proximal activation events. In contrast to the observed Ag-specific hyporesponsiveness, the Ly-6A.2 transgenic CD4+ T cells produced IL-4 independent of the interactions between Ly-6A.2 and the candidate Ly-6A.2 ligand. Our results suggest that 1) interaction of Ly-6A.2 with a candidate ligand regulates clonal expansion of CD4+ Th cells in response to an Ag (these results also provide further functional evidence for presence of Ly-6A.2 ligand on APC); and 2) Ly-6A.2 expression on CD4+ T cells promotes production of IL-4, a Th2 differentiation factor.
Low-Affinity Anti-Smith Antigen B Cells Are Regulated by Anergy as Opposed to Developmental Arrest or Differentiation to B-1
Authors: Borrero Michelle , Clarke Stephen H.
Published: April 2014

Understanding the regulation of B lymphocytes specific for self-Ags targeted in human and murine systemic lupus erythematosus, such as the ribonucleoprotein Smith Ag (Sm), is crucial to understanding the etiology of this autoimmune disease. To address the role of B cell receptor affinity in the regulation of anti-Sm B cells, we generated low-affinity anti-Sm transgenic mice by combining the anti-Sm 2-12H transgene with a Vκ8 transgene. In contrast to 2-12H transgenic mice, in which anti-Sm B cells are predominantly splenic transitional, and peritoneal B-1, low-affinity anti-Sm B cells are long-lived B-2 cells and are found in the spleen, lymph nodes, and peritoneum. However, they are unresponsive to LPS in vitro, indicating that they are anergic, although they do not down-regulate IgM and are not excluded from follicles even in the presence of nonautoreactive B cells. Thus, low-affinity anti-Sm B cells appear to have a partial form of anergy. Interestingly, these cells have elevated levels of MHC class II and CD95, but not CD40, CD80, or CD86, suggesting that they are poised to undergo deletion rather than activation upon T cell encounter. These data identify anergy as a mechanism involved in anti-Sm B cell regulation.