bluuebunny/crossref_metadata_2025 huggingface dataset:
Prolonged Production of TNF-α Exacerbates Illness during Respiratory Syncytial Virus Infection
Authors: Rutigliano John A. , Graham Barney S.
Published: April 2014

CD8+ CTL are the main effector cells responsible for resolving viral infections. However, the CTL response to respiratory syncytial virus (RSV) infection in mice facilitates viral clearance at the expense of significant immunopathology. Previous reports have shown a strong correlation between the mechanism of CTL activity and the severity of RSV-induced illness. Furthermore, experiments in perforin knockout mice revealed that antiviral cytokine production temporally correlated with RSV-induced illness. In the current study, we show that TNF-α is the dominant mediator of RSV-associated illness, and it is also important for clearance of virus-infected cells during the early stages of infection. We also demonstrate that IFN-γ plays a protective role in conjunction with perforin/granzyme-mediated killing. Preliminary experiments in gld mice that express nonfunctional Fas ligand (FasL) revealed that RSV-induced illness is significantly reduced in the absence of FasL-mediated killing. Antiviral cytokine production was not elevated in the absence of FasL, suggesting a possible link between FasL and antiviral cytokine activity. This work shows that multiple phenotypic subsets of CD8+ CTLs respond to RSV infection, each with varying capacities for clearance of virus-infected cells and the induction of illness. In addition, the revelation that TNF-α is the principal mediator of RSV-induced illness means that administration of TNF receptor antagonists, in combination with antiviral therapy, may be an effective method to treat RSV infections.
Expression of a Functional C5a Receptor in Regenerating Hepatocytes and Its Involvement in a Proliferative Signaling Pathway in Rat
Authors: Daveau Maryvonne , Benard Magalie , Scotte Michel , Schouft Marie-Therese , Hiron Martine , Francois Arnaud , Salier Jean-Philippe , Fontaine Marc
Published: April 2014

Activation of the complement system generates the anaphylatoxin C5a whose activities are mediated through its binding to the widely expressed C5aR. C5aR mRNA and protein expressions are known to be induced in rat hepatocytes under inflammatory conditions. However, little is known about the role of the C5a/C5aR complex in liver and its involvement during a proliferative process. We have evaluated the expression of C5aR in regenerating rat hepatocytes following a partial hepatectomy and in hepatocyte cultures. C5aR induction was observed in hepatocytes from regenerating liver, as well as in normal hepatocytes under a culture-induced stress. The effect of a stimulation by a C5a agonist upon the synthesis of a growth factor/receptor pair (hepatocyte growth factor/c-Met) was also evaluated. Our data demonstrated an up-regulated expression of hepatocyte growth factor and c-Met mRNAs, but we failed to observe a direct mitogenic effect of C5a in culture. However, a significantly increased expression of cyclin E and D1mRNA levels, as well as an increased BrdU incorporation, were observed in rats given an i.v. C5a agonist injection following an 80% partial hepatectomy. These studies demonstrate for the first time that: 1) C5aR is up-regulated during liver regeneration, 2) the binding of C5a to C5aR promotes a growth response, and 3) C5aR is involved in a cell cycle signaling pathway. Taken together, these findings point to a novel role for the hepatic C5aR implicating this complement system in the context of normal or abnormal proliferative pathways.
C5a Initiates the Inflammatory Cascade in Immune Complex Peritonitis
Authors: Godau Jeanne , Heller Tanja , Hawlisch Heiko , Trappe Matthew , Howells Elaine , Best Jennifer , Zwirner Jörg , Verbeek J. Sjef , Hogarth P. Mark , Gerard Craig , van Rooijen Nico , Klos Andreas , Gessner J. Engelbert , Köhl Jörg
Published: April 2014

Immune complex (IC)-induced inflammation is integral to the pathogenesis of several autoimmune diseases. ICs activate the complement system and interact with IgG FcγR. In this study, we demonstrate that activation of the complement system, specifically generation of C5a, initiates the neutrophilic inflammation in IC peritonitis. We show that ablation of C5a receptor signaling abrogates neutrophil recruitment in wild-type mice and prevents the enhancement of neutrophil migration seen in FcγRIIB−/− mice, suggesting that C5aR signaling is the crucial initial event upstream of FcγR signaling. We also provide evidence that C5a initiates the inflammatory cascade both directly, through C5aR-mediated effector functions on infiltrating and resident peritoneal cells, and indirectly, through shifting the balance between activating and inhibitory FcγRs on resident cells toward an inflammatory phenotype. We conclude that complement activation and C5a generation are prerequisites for IC-induced inflammation through activating FcγR, which amplifies complement-induced inflammation in autoimmunity.
Ig-Reactive CD4+CD25+ T Cells from Tolerized (New Zealand Black × New Zealand White)F1 Mice Suppress In Vitro Production of Antibodies to DNA
Authors: La Cava Antonio , Ebling Fanny M. , Hahn Bevra H.
Published: April 2014

We have recently shown that tolerogenic administration of an artificial peptide (pConsensus) that is based on sequences within the VH regions of several murine anti-dsDNA Ig delays appearance of autoantibodies in female (New Zealand Black (NZB) × New Zealand White (NZW))F1 (NZB/W F1) mice and significantly prolongs their survival. The aim of this study was to characterize the T cell population(s) involved in pConsensus-induced down-regulation of autoimmune responses in tolerized NZB/W F1 mice. Using MHC class II dimers loaded with tolerogenic peptide, we found that pCons favored expansion of peptide-reactive CD4+CD25+ regulatory T cells (TR) that inhibited in vitro production of anti-dsDNA Ab-forming cells. Suppression by TR was abrogated by the presence in culture of Ab to glucocorticoid-induced TNFR family member 18 or to TGFβ latency-associated protein. These findings suggest possible relevance of Ag specificity in the mechanism of TR-mediated immune tolerance to Ig-derived peptides in NZB/W F1 mice.
Frequent Joining of Bcl-2 to a JH6 Gene in Hepatitis C Virus-Associated t(14;18)
Authors: Sasso Eric H. , Martinez Marina , Yarfitz Stuart L. , Ghillani Pascale , Musset Lucile , Piette Jean-Charles , Cacoub Patrice
Published: April 2014

The t(14;18) chromosomal translocation, which joins the Bcl-2 proto-oncogene to an Ig JH gene, has increased prevalence in patients chronically infected with hepatitis C virus (HCV). We now establish a link between the molecular structure and clinical occurrence of HCV-associated t(14;18). A t(14;18) was detected by PCR in leukocytes from 22 of 46 HCV-infected patients (48%) and 11 of 54 healthy controls (20%) (p = 0.0053). Nucleotide sequence analysis of the Bcl-2/JH joins found a JH6 gene in 18 of 22 (82%) t(14;18) from HCV+ patients, and 3 of 8 (38%) from controls (p = 0.031). The t(14;18) rarely contained JH gene mutations, or an intervening region sequence suggestive of D gene rearrangement or templated nucleotide insertion. Analysis of published t(14;18) nucleotide sequences established that the JH6 prevalence in t(14;18) from normal/nonneoplastic controls (48%) was significantly lower than in t(14;18) from our HCV+ patients (p = 0.004) or from non-Hodgkin’s lymphomas (66%, p = 0.003). We conclude that the increased prevalence of t(14;18) in HCV+ patients occurs with a strong bias for Bcl-2/JH6 joins. In this regard, HCV-associated t(14;18) more closely resemble t(14;18) in lymphomas than t(14;18) from normal subjects.
Stromelysin-2 (Matrix Metalloproteinase 10) Is Inducible in Lymphoma Cells and Accelerates the Growth of Lymphoid Tumors In Vivo
Authors: Van Themsche Céline , Alain Tommy , Kossakowska Anna E. , Urbanski Stefan , Potworowski Édouard F. , St-Pierre Yves
Published: April 2014

Matrix metalloproteinase (MMP) 10 (stromelysin-2) is known to degrade various components of the extracellular matrix; however, the signals that regulate its expression and its role in lymphoma growth remain unknown. In the present work, we report the up-regulated expression of MMP10 in T lymphoma cells following contact with endothelial cells. The induction of MMP10 was found to be dependent on the specific interaction between LFA-1 and ICAM-1, which play a central role in regulating the expression of genes involved in the rate-limiting steps of lymphoma development. MMP10, but not MMP3 (stromelysin-1), was also up-regulated in human B lymphoma cells following exposure to IL-4, IL-6, and IL-13, but not to IL-1. To gain further insight into the role of MMP10 in lymphoma development, we generated lymphoma cell lines constitutively expressing high levels of MMP10 and studied these cells for their ability to form thymic lymphoma in vivo. Mice injected with lymphoma cells constitutively expressing MMP10 developed thymic lymphoma more rapidly than those injected with control lymphoma cells. These results provide the first in vivo evidence that overexpression of MMP10 promotes tumor development, and indicate that MMP10 induction is an important pathway activated not only upon ICAM-1/LFA-1-mediated intercellular contact, but also following activation of tumor cells with inflammatory cytokines.
Immunological Synapse Formation Licenses CD40-CD40L Accumulations at T-APC Contact Sites
Authors: Boisvert Judie , Edmondson Samuel , Krummel Matthew F.
Published: April 2014

The maintenance of tolerance is likely to rely on the ability of a T cell to polarize surface molecules providing “help” to only specific APCs. The formation of a mature immunological synapse leads to concentration of the TCR at the APC interface. In this study, we show that the CD40-CD154 receptor-ligand pair is also highly concentrated into a central region of the synapse on mouse lymphocytes only after the formation of the TCR/CD3 c-SMAC. Concentration of this ligand was strictly dependent on TCR recognition, the binding of ICAM-1 to T cell integrins and the presence of an intact cytoskeleton in the T cells. This may provide a novel explanation for the specificity of T cell help directing the help signal to the site of Ag receptor signal. It may also serve as a site for these molecular aggregates to coassociate and/or internalize alongside other signaling receptors.
LFA-1 Contributes an Early Signal for NK Cell Cytotoxicity
Authors: Barber Domingo F. , Faure Mathias , Long Eric O.
Published: April 2014

Cytotoxicity of human NK cells is activated by receptors that bind ligands on target cells, but the relative contribution of the many different activating and inhibitory NK cell receptors is difficult to assess. In this study, we describe an experimental system that circumvents some of the difficulties. Adhesion through β2 integrin LFA-1 is a common requirement of CTLs and NK cells for efficient lysis of target cells. However, the contribution of LFA-1 to activation signals for NK cell cytotoxicity, besides its role in adhesion, is unclear. The role of LFA-1 was evaluated by exposing NK cells to human ICAM-1 that was either expressed on a Drosophila insect cell line, or directly coupled to beads. Expression of ICAM-1 on insect cells was sufficient to induce lysis by NK cells through LFA-1. Coexpression of peptide-loaded HLA-C with ICAM-1 on insect cells blocked the LFA-1-dependent cytotoxicity of NK cells that expressed HLA-C-specific inhibitory receptors. Polarization of cytotoxic granules in NK cells toward ICAM-1- and ICAM-2-coated beads showed that engagement of LFA-1 alone is sufficient to initiate activation signals in NK cells. Thus, in contrast to T cells, in which even adhesion through LFA-1 is dependent on signals from other receptors, NK cells receive early activation signals directly through LFA-1.
CC Chemokine Ligands 25 and 28 Play Essential Roles in Intestinal Extravasation of IgA Antibody-Secreting Cells
Authors: Hieshima Kunio , Kawasaki Yuri , Hanamoto Hitoshi , Nakayama Takashi , Nagakubo Daisuke , Kanamaru Akihisa , Yoshie Osamu
Published: April 2014

CCL25 (also known as thymus-expressed chemokine) and CCL28 (also known as mucosae-associated epithelial chemokine) play important roles in mucosal immunity by recruiting IgA Ab-secreting cells (ASCs) into mucosal lamina propria. However, their exact roles in vivo still remain to be defined. In this study, we first demonstrated in mice that IgA ASCs in small intestine expressed CCR9, CCR10, and CXCR4 on the cell surface and migrated to their respective ligands CCL25, CCL28, and CXCL12 (also known as stromal cell-derived factor 1), whereas IgA ASCs in colon mainly expressed CCR10 and CXCR4 and migrated to CCL28 and CXCL12. Reciprocally, the epithelial cells of small intestine were immunologically positive for CCL25 and CCL28, whereas those of colon were positive for CCL28 and CXCL12. Furthermore, the venular endothelial cells in small intestine were positive for CCL25 and CCL28, whereas those in colon were positive for CCL28, suggesting their direct roles in extravasation of IgA ASCs. Consistently, in mice orally immunized with cholera toxin (CT), anti-CCL25 suppressed homing of CT-specific IgA ASCs into small intestine, whereas anti-CCL28 suppressed homing of CT-specific IgA ASCs into both small intestine and colon. Reciprocally, CT-specific ASCs and IgA titers in the blood were increased in mice treated with anti-CCL25 or anti-CCL28. Anti-CXCL12 had no such effects. Finally, both CCL25 and CCL28 were capable of enhancing α4 integrin-dependent adhesion of IgA ASCs to mucosal addressin cell adhesion molecule-1 and VCAM-1. Collectively, CCL25 and CCL28 play essential roles in intestinal homing of IgA ASCs primarily by mediating their extravasation into intestinal lamina propria.
Stimulatory Killer Ig-Like Receptors Modulate T Cell Activation through DAP12-Dependent and DAP12-Independent Mechanisms
Authors: Snyder Melissa R. , Nakajima Takako , Leibson Paul J. , Weyand Cornelia M. , Goronzy Jörg J.
Published: April 2014

Stimulatory killer Ig-like receptors (KIRs) are expressed by various lymphocytes, including NK cells and subsets of T cells. In NK cells, KIRs associate with the adapter molecule KARAP/DAP12, which confers the ability to function as an independent activation unit. The function of KIRs and killer cell activating receptor-associated protein (KARAP)/DAP12 in T cells is unclear. By flow cytometry, we demonstrated that CD4+CD28null T cells heterogeneously express KIRs and/or KARAP/DAP12. In clones that lacked expression of KARAP/DAP12, the stimulatory KIR KIR2DS2 signaled through the JNK pathway, but did not activate the ERK pathway. However, in the presence of KARAP/DAP12, stimulation through KIR2DS2 led to phosphorylation of both JNK and ERK. Transfection experiments confirmed that KIR2DS2-mediated ERK phosphorylation was dependent on KARAP/DAP12. The differential signaling of KIR2DS2 through association with alternative adapter molecules resulted in differential regulation of cellular activity. In clones that lacked expression of KARAP/DAP12, stimulation of KIR2DS2 did not induce cytotoxicity. However, KIR2DS2 did augment suboptimal TCR stimulation, leading to enhanced IFN-γ production. In clones that expressed KARAP/DAP12, KIR2DS2 directly activated both cytotoxicity and IFN-γ production without the need for TCR-derived signals. The function of stimulatory KIRs in T cells is determined by the expression of the appropriate adapter molecule. Expression of KARAP/DAP12 is sufficient to convert a costimulatory KIR into a stimulatory molecule. These differing functions mediated by alternative signaling pathways have implications for the pathogenesis of diseases such as rheumatoid arthritis and acute coronary syndromes, in which aberrant expression of KIRs on T cells is frequently observed.
Intratumoral CC Chemokine Ligand 5 Overexpression Delays Tumor Growth and Increases Tumor Cell Infiltration
Authors: Lavergne Elise , Combadière Christophe , Iga Mutsunori , Boissonnas Alexandre , Bonduelle Olivia , Maho Maud , Debré Patrice , Combadiere Behazine
Published: April 2014

Chemokines participate in the antitumor immune response by regulating the movement and positioning of lymphocytes as well as effector functions and may thus be candidates for use in antitumor therapy. To test whether CCL5, a chemokine involved in the recruitment of a wide spectrum of immunocompetent cells, can control tumor growth, we forced its expression at mouse tumor sites. Tumor growth was reduced in mice with s.c. syngeneic CCL5-EL-4 compared with EL-4-injected mice, whereas both reduced tumor growth and incidence were observed in mice with OVA-expressing EG-7 transfected with CCL5 compared with EG-7-injected mice. Significant antitumor effects were observed soon after intratumoral injection of DNA plasmid coding for chimeric CCL5-Ig. Importantly, quantitative RT-PCR assays showed that the amount of CCL5 expression at the tumor site determined the effectiveness of the antitumor response, which was associated with infiltration of increased numbers of NK, CD4, and CD8 cells at the tumor site. This effect was lost in mice deficient for T/B lymphocytes (RAG-2 knockout) or for CCR5 (CCR5 knockout). Together, these data demonstrate the antitumor activity of intratumoral CCL5 overexpression, due to its recruitment of immunocompetent cells, and the potential usefulness of chimeric CCL5-Ig DNA as an agent in cancer therapy.
Discrete Event Modeling of CD4+ Memory T Cell Generation
Authors: Zand Martin S. , Briggs Benjamin J. , Bose Anirban , Vo Thuong
Published: April 2014

Studies of memory T cell differentiation are hampered by a lack of quantitative models to test hypotheses in silico before in vivo experimentation. We created a stochastic computer model of CD4+ memory T cell generation that can simulate and track 101–108 individual lymphocytes over time. Parameters for the model were derived from experimental data using naive human CD4+ T cells stimulated in vitro. Using discrete event computer simulation, we identified two key variables that heavily influence effector burst size and the persistent memory pool size: the cell cycle dependent probability of apoptosis, and the postactivation mitosis at which memory T cells emerge. Multiple simulations were performed and varying critical parameters permitted estimates of how sensitive the model was to changes in all of the model parameters. We then compared two hypotheses of CD4+ memory T cell generation: maturation from activated naive to effector to memory cells (model I) vs direct progression from activated naive to memory cells (model II). We find that direct progression of naive to memory T cells does not explain published measurements of the memory cell mass unless postactivation expansion of the memory cell cohort occurs. We conclude that current models suggesting direct progression of activated naive cells to the persistent memory phenotype (model II) do not account for the experimentally measured size of the postactivation CD4+, Ag-specific, memory T cell cohort.
Sphingosine Contributes to Glucocorticoid-Induced Apoptosis of Thymocytes Independently of the Mitochondrial Pathway
Authors: Lépine Sandrine , Lakatos Boris , Courageot Marie-Pierre , Le Stunff Hervé , Sulpice Jean-Claude , Giraud Françoise
Published: April 2014

During the selection process in the thymus, most thymocytes are eliminated by apoptosis through signaling via TCR or glucocorticoids. The involvement of ceramide (Cer) and sphingosine (SP), important apoptotic mediators, remains poorly defined in glucocorticoid-induced apoptosis. We report that, in mouse thymocytes, apoptosis triggered by 10−6 M dexamethasone (DX) was preceded by a caspase-dependent Cer and SP generation, together with activation of acidic and neutral ceramidases. Apoptosis was drastically reduced by blocking either sphingolipid production (by acid sphingomyelinase inhibitor) or SP production (by ceramidase inhibitors), but not by inhibition of de novo Cer synthesis. Thus, SP generated through acid sphingomyelinase and ceramidase activity would contribute to the apoptotic effect of DX. Consistent with this hypothesis, SP addition or inhibition of SP kinase induced thymocyte apoptosis. DX induced a proteasome-dependent loss of mitochondrial membrane potential (Δψm) and caspase-8, -3, and -9 processing. Apoptosis was abolished by inhibition of Δψm loss or caspase-8 or -3, but not caspase-9. Δψm loss was independent of SP production and caspase-8, -3, and -9 activation. However, inhibition of SP production reduced caspase-8 and -3, but not caspase-9 processing. Proteasome inhibition impaired activation of the three caspases, whereas inhibition of Δψm loss solely blocked caspase-9 activation. These data indicate that DX-induced apoptosis is mediated in part by SP, which contributes, together with proteasome activity, to caspase-8-3 processing independently of mitochondria, and in part by the proteasome/mitochondria pathway, although independently of caspase-9 activation.
CD8 T Cell Responses to Lymphocytic Choriomeningitis Virus in Early Growth Response Gene 1-Deficient Mice
Authors: Singh Anju , Svaren John , Grayson Jason , Suresh M.
Published: April 2014

Previous in vitro work has implicated a role for transcriptional factor early growth response gene 1 (EGR1) in regulating immune responses. However, the in vivo role of EGR1 in orchestrating T cell responses has not been studied. To investigate the importance of EGR1 in T cell immunity, we compared Ag-specific CD8 T cell responses between wild type (+/+) and EGR1-deficient (EGR1−/−) mice following an acute infection with lymphocytic choriomeningitis virus (LCMV). These studies revealed that the expansion of LCMV-specific CD8 T cells was substantially reduced in EGR1−/− mice, as compared with +/+ mice. The reduced numbers of LCMV-specific CD8 T cells in EGR1−/− mice were not due to an intrinsic T cell defect per se because purified EGR1-deficient T cells exhibited normal proliferative response to anti-CD3 stimulation in vitro, and underwent normal activation and expansion in response to LCMV upon adoptive transfer into T cell-deficient mice. Furthermore, adoptive transfer of CD8 T cells bearing a transgenic TCR into EGR1−/− mice showed that EGR1 deficiency in non-CD8 T cells impaired CD8 T cell expansion in vivo following an LCMV infection. Further investigations on accessory cells showed that bone marrow-derived dendritic cells from EGR1−/− mice did not exhibit detectable impairment to prime Ag-specific CD8 T cell responses in vivo. However, in LCMV-infected mice, EGR1 deficiency selectively impaired the maturation of CD8α+ve plasmacytoid dendritic cells. Taken together, our findings suggest that EGR1 might promote expansion of CD8 T cells during an acute viral infection by modulating the cues in the lymphoid microenvironment.
Dual Function of the Extracellular Matrix: Stimulatory for Cell Cycle Progression of Naive T Cells and Antiapoptotic for Tissue-Derived Memory T Cells
Authors: Sturm Andreas , Krivacic Kimberley A. , Fiocchi Claudio , Levine Alan D.
Published: April 2014

Tissue T cells encounter Ag in a distinct microenvironment, where they are embedded in the interstitial extracellular matrix (ECM). In contrast, while naive T cells are exposed to Ag in the lymph node, immediately after naive T cells are activated they must extravasate into the ECM to function effectively. Because integrin-mediated adhesion to the ECM modulates cell cycle progression and survival in adherent nonimmune cells, we hypothesize that blood and tissue-derived T cells have similarly adapted their behavior to their first or continued encounter with ECM. T cells from peripheral blood (PBT) and tissue (the intestinal lamina propria T cell (LPT)) were stimulated with anti-CD3-coated beads in the presence or absence of native ECM derived from intestinal fibroblasts, plate-immobilized fibronectin, or collagen type I. Native ECM and collagen, but not fibronectin, induced in anti-CD3 activated PBT a 4- to 5-fold increase in the entry, progression, and completion of the cell cycle over that triggered by anti-CD3 alone. Neutralizing β1 integrin Abs abrogated this increase. None of these ECM proteins stimulated cell cycle progression in LPT. In contrast, anti-CD3 activation of LPT in the presence of native ECM and fibronectin reduced activation-induced cell death by 40%. These results demonstrate that naive and effector/memory T cells respond differently upon exposure to specific ECM components. When naive PBT encounter Ag in the context of ECM, their progression through the cell cycle is enhanced, favoring clonal expansion; while tissue T cell longevity may be mediated by interactions with the ECM.
Immunoproteasomes Down-Regulate Presentation of a Subdominant T Cell Epitope from Lymphocytic Choriomeningitis Virus
Authors: Basler Michael , Youhnovski Nikolay , van den Broek Maries , Przybylski Michael , Groettrup Marcus
Published: April 2014

The cytotoxic T cell response to pathogens is usually directed against a few immunodominant epitopes, while other potential epitopes are either subdominant or not used at all. In C57BL/6 mice, the acute cytotoxic T cell response against lymphocytic choriomeningitis virus is directed against immunodominant epitopes derived from the glycoprotein (gp33–41) and the nucleoprotein (NP396–404), while the gp276–286 epitope remains subdominant. Despite extensive investigations, the reason for this hierarchy between epitopes is not clear. In this study, we show that the treatment of cells with IFN-γ enhanced the presentation of gp33–41, whereas presentation of the gp276–286 epitope from the same glycoprotein was markedly reduced. Because proteasomes are crucially involved in epitope generation and because IFN-γ treatment in vitro and lymphocytic choriomeningitis virus infection in vivo lead to a gradual replacement of constitutive proteasomes by immunoproteasomes, we investigated the role of proteasome composition on epitope hierarchy. Overexpression of the active site subunits of immunoproteasomes LMP2, LMP7, and MECL-1 as well as overexpression of LMP2 alone suppressed the presentation of the gp276–286 epitope. The ability to generate gp276–286-specific CTLs was enhanced in LMP2- and LMP7-deficient mice, and macrophages from these mice showed an elevated presentation of this epitope. In vitro digests demonstrated that fragmentation by immunoproteasomes, but not constitutive proteasomes led to a preferential destruction of the gp276 epitope. Taken together, we show that LMP2 and LMP7 can at least in part determine subdominance and shape the epitope hierarchy of CTL responses in vivo.
The Murine NK Receptor 2B4 (CD244) Exhibits Inhibitory Function Independent of Signaling Lymphocytic Activation Molecule-Associated Protein Expression
Authors: Mooney Jill M. , Klem Jennifer , Wülfing Christoph , Mijares Lilia A. , Schwartzberg Pamela L. , Bennett Michael , Schatzle John D.
Published: April 2014

2B4 (CD244) is a receptor belonging to the CD2-signaling lymphocytic activation molecule family and is found on all murine NK cells and a subset of NKT and CD8+ T cells. Murine 2B4 is expressed as two isoforms (2B4 short and 2B4 long) that arise by alternative splicing. They differ only in their cytoplasmic domains and exhibit opposing function when expressed in the RNK-16 cell line. The ligand for 2B4, CD48, is expressed on all hemopoietic cells. Previous studies have shown that treatment of NK cells with a 2B4 mAb results in increased cytotoxicity and IFN-γ production. In this report, we used CD48+/− variants of the P815 tumor cell line and 2B4 knockout mice to show that engagement of 2B4 by its counterreceptor, CD48, expressed on target cells leads to an inhibition in NK cytotoxicity. The addition of 2B4 or CD48 mAb relieves this inhibition resulting in enhanced target cell lysis. This 2B4-mediated inhibition acts independently of signaling lymphocytic activation molecule-associated protein expression. Imaging studies show that 2B4 preferentially accumulates at the interface between NK and target cells during nonlytic events also indicative of an inhibitory receptor. This predominant inhibitory function of murine 2B4 correlates with increased 2B4 long isoform level expression over 2B4 short.
Histone Deacetylase 3, a Class I Histone Deacetylase, Suppresses MAPK11-Mediated Activating Transcription Factor-2 Activation and Represses TNF Gene Expression
Authors: Mahlknecht Ulrich , Will Jutta , Varin Audrey , Hoelzer Dieter , Herbein Georges
Published: April 2014

During inflammatory events, the induction of immediate-early genes, such as TNF-α, is regulated by signaling cascades including the JAK/STAT, NF-κB, and the p38 MAPK pathways, which result in phosphorylation-dependent activation of transcription factors. We observed the direct interaction of histone deacetylase (HDAC) 3, a class I histone deacetylase, with MAPK11 (p38 β isoform) by West-Western-based screening analysis, pull-down assay, and two-hybrid system analysis. Results further indicated that HDAC3 decreases the MAPK11 phosphorylation state and inhibits the activity of the MAPK11-dependent transcription factor, activating transcription factor-2 (ATF-2). LPS-mediated activation of ATF-2 was inhibited by HDAC3 in a time- and dose-dependent manner. Inhibition of HDAC3 expression by RNA interference resulted in increased ATF-2 activation in response to LPS stimulation. In agreement with decreased ATF-2 transcriptional activity by HDAC3, HDAC3-repressed TNF gene expression, and TNF protein production observed in response to LPS stimulation. Therefore, our results indicate that HDAC3 interacts directly and selectively with MAPK11, represses ATF-2 transcriptional activity, and acts as a regulator of TNF gene expression in LPS-stimulated cells, especially in mononuclear phagocytes.
A Novel Mutation in IFN-γ Receptor 2 with Dominant Negative Activity: Biological Consequences of Homozygous and Heterozygous States
Authors: Rosenzweig Sergio D. , Dorman Susan E. , Uzel Gulbu , Shaw Stephen , Scurlock Amy , Brown Margaret R. , Buckley Rebecca H. , Holland Steven M.
Published: April 2014

We identified two siblings homozygous for a single base pair deletion in the IFN-γR2 transmembrane domain (791delG) who presented with multifocal Mycobacterium abscessus osteomyelitis (patient 1) and disseminated CMV and Mycobacterium avium complex infection (patient 2), respectively. Although the patients showed no IFN-γR activity, their healthy heterozygous parents showed only partial IFN-γR activity. An HLA-identical bone marrow transplant from the mother led patient 1 to complete hemopoietic reconstitution, but only partial IFN-γR function. We cloned and expressed fluorescent fusion proteins of the wild-type IFN-γR2, an IFN-γR2 mutant previously described to produce a complete autosomal recessive deficiency (278del2), and of 791delG to determine whether the intermediate phenotype in the 791delG heterozygous state was caused by haploinsufficiency or a dominant negative effect. When cotransfected together with the wild-type vector into IFN-γR2-deficient fibroblasts, the fusion protein with 791delG inhibited IFN-γR function by 48.7 ± 5%, whereas fusion proteins with 278del2 had no inhibitory effect. Confocal microscopy of 791delG fusion proteins showed aberrant diffuse intracellular accumulation without plasma membrane localization. The fusion protein created by 791delG did not complete Golgi processing, and was neither expressed on the plasma membrane, nor shed extracellularly. The mutant construct 791delG exerts dominant negative effects on IFN-γ signaling without cell surface display, suggesting that it is acting on pathways other than those involved in cell surface recognition of ligand.
Identification of Immunodominant Sites on the Spike Protein of Severe Acute Respiratory Syndrome (SARS) Coronavirus: Implication for Developing SARS Diagnostics and Vaccines
Authors: He Yuxian , Zhou Yusen , Wu Hao , Luo Baojun , Chen Jingming , Li Wanbo , Jiang Shibo
Published: April 2014

The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is not only responsible for receptor binding and virus fusion, but also a major Ag among the SARS-CoV proteins that induces protective Ab responses. In this study, we showed that the S protein of SARS-CoV is highly immunogenic during infection and immunizations, and contains five linear immunodominant sites (sites I to V) as determined by Pepscan analysis with a set of synthetic peptides overlapping the entire S protein sequence against the convalescent sera from SARS patients and antisera from small animals immunized with inactivated SARS-CoV. Site IV located in the middle region of the S protein (residues 528–635) is a major immunodominant epitope. The synthetic peptide S603–634, which overlaps the site IV sequence reacted with all the convalescent sera from 42 SARS patient, but none of the 30 serum samples from healthy blood donors, suggesting its potential application as an Ag for developing SARS diagnostics. This study also provides information useful for designing SARS vaccines and understanding the SARS pathogenesis.
SalmonellaEscape from Antigen Presentation Can Be Overcome by Targeting Bacteria to Fcγ Receptors on Dendritic Cells
Authors: Tobar Jaime A. , González Pablo A. , Kalergis Alexis M.
Published: April 2014

Dendritic cells (DCs) are professional APCs with the unique ability to activate naive T cells, which is required for initiation of the adaptive immune response against pathogens. Therefore, interfering with DC function would be advantageous for pathogen survival and dissemination. In this study we provide evidence suggesting that Salmonella enterica serovar typhimurium, the causative agent of typhoid disease in the mouse, interferes with DC function. Our results indicate that by avoiding lysosomal degradation, S. typhimurium impairs the ability of DCs to present bacterial Ags on MHC class I and II molecules to T cells. This process could correspond to a novel mechanism developed by this pathogen to evade adaptive immunity. In contrast, when S. typhimurium is targeted to FcγRs on DCs by coating bacteria with Salmonella-specific IgG, bacterial Ags are efficiently processed and presented on MHC class I and class II molecules. This enhanced Ag presentation leads to a robust activation of bacteria-specific T cells. Laser confocal microscopy experiments show that virulent S. typhimurium is rerouted to the lysosomal degradation pathway of DCs when internalized through FcγR. These observations are supported by electron microscopy studies demonstrating that internalized S. typhimurium shows degradation signs only when coated with IgG and captured by FcγRs on DCs. Therefore, our data support a potential role for bacteria-specific IgG on the augmentation of Ag processing and presentation by DCs to T cells during the immune response against intracellular bacteria.
Herpes Simplex Virus Type-1-Induced Activation of Myeloid Dendritic Cells: The Roles of Virus Cell Interaction and Paracrine Type I IFN Secretion
Authors: Pollara Gabriele , Jones Meleri , Handley Matthew E. , Rajpopat Mansi , Kwan Antonia , Coffin Robert S. , Foster Graham , Chain Benjamin , Katz David R.
Published: April 2014

Adaptive cellular immunity is required to clear HSV-1 infection in the periphery. Myeloid dendritic cells (DCs) are the first professional Ag-presenting cell to encounter the virus after primary and secondary infection and thus the consequences of their infection are important in understanding the pathogenesis of the disease and the response to the virus. Following HSV-1 infection, both uninfected and infected human DCs acquire a more mature phenotype. In this study, we demonstrate that type I IFN secreted from myeloid DC mediates bystander activation of the uninfected DCs. Furthermore, we confirm that this IFN primes DCs for elevated IL-12 p40 and p70 secretion. However, secretion of IFN is not responsible for the acquisition of a mature phenotype by HSV-1-infected DC. Rather, virus binding to a receptor on the cell surface induces DC maturation directly, through activation of the NF-κB and p38 MAPK pathways. The binding of HSV glycoprotein D is critical to the acquisition of a mature phenotype and type I IFN secretion. The data therefore demonstrate that DCs can respond to HSV exposure directly through recognition of viral envelope structures. In the context of natural HSV infection, the coupling of viral entry to the activation of DC signaling pathways is likely to be counterbalanced by viral disruption of DC maturation. However, the parallel release of type I IFN may result in paracrine activation so that the DCs are nonetheless able to mount an adaptive immune response.
The Essential Role of the Intestinal Microbiota in Facilitating Acute Inflammatory Responses
Authors: Souza Danielle G. , Vieira Angélica T. , Soares Adriana C. , Pinho Vanessa , Nicoli Jacques R. , Vieira Leda Q. , Teixeira Mauro M.
Published: April 2014

The restoration of blood flow, i.e., reperfusion, is the treatment of choice to save viable tissue following acute ischemia of a vascular territory. Nevertheless, reperfusion can be accompanied by significant inflammatory events that limit the beneficial effects of blood flow restoration. To evaluate the potential role of the intestinal microbiota in facilitating the development of tissue injury and systemic inflammation, germ-free and conventional mice were compared in their ability to respond to ischemia and reperfusion injury. In conventional mice, there was marked local (intestine) and remote (lung) edema formation, neutrophil influx, hemorrhage, and production of TNF-α, KC, MIP-2, and MCP-1. Moreover, there was an increase in the concentration of serum TNF-α and 100% lethality. In germ-free mice, there was no local, remote, or systemic inflammatory response or lethality after intestinal ischemia and reperfusion and, in contrast to conventional mice, germ-free animals produced greater amounts of IL-10. Similar results were obtained after administration of LPS, i.e., little production of TNF-α or lethality and production of IL-10 after LPS in germ-free mice. Blockade of IL-10 with Abs induced marked inflammation and lethality in germ-free mice after ischemia and reperfusion or LPS administration, demonstrating that the ability of these mice to produce IL-10 was largely responsible for their “no inflammation” phenotype. This was consistent with the prevention of reperfusion-associated injury by the exogenous administration of IL-10 to conventional mice. Thus, the lack of intestinal microbiota is accompanied by a state of active IL-10-mediated inflammatory hyporesponsiveness.
Receptor Editing in Positive and Negative Selection of B Lymphopoiesis
Authors: Edry Efrat , Melamed Doron
Published: April 2014

In B lymphopoiesis, Ag receptor expression and signaling are critical to determine developmental progression, survival, and activation. Several positive and negative selection checkpoints to test this receptor have been described in B lymphopoiesis, aiming to ensure the generation of functionally competent, nonautoimmune repertoire. Secondary Ag receptor gene recombination allows B lymphocytes to replace an inappropriate receptor with a new receptor, a mechanism called receptor editing. This salvage mechanism uncouples the Ag receptor fate from that of the cell itself, suggesting that B cell repertoire is regulated by a process of receptor selection. Secondary rearrangements are stimulated in different stages of B cell development, where editing of the receptor is necessary to fulfill stage-specific requirements. In this study, we discuss the contribution of receptor editing in B lymphopoiesis and its regulation by positive and negative selection signals.
Dissection of BXSB Lupus Phenotype Using Mice Congenic for Chromosome 1 Demonstrates That Separate Intervals Direct Different Aspects of Disease
Authors: Haywood Michelle E. K. , Rogers Nicola J. , Rose S. Jane , Boyle Joseph , McDermott Aileen , Rankin Joanna M. , Thiruudaian Vasuky , Lewis Margarita R. , Fossati-Jimack Liliane , Izui Shozo , Walport Mark J. , Morley Bernard J.
Published: April 2014

To dissect the individual effects of the four non-MHC, autosomal loci (Bxs1 to Bxs4) that contribute to SLE susceptibility in BXSB mice, we generated congenic lines from chromosome 1 on a C57BL/10.YBXSB (B10.Yaa) background for the intervals (values in megabases (Mb)) Bxs1 (46.3-89.2 Mb), Bxs1/4 (20.0-65.9 Mb), Bxs1/2 (64.4-159.0 Mb), and Bxs2/3 (105.4-189.0 Mb). Glomerulonephritis, qualitatively similar to that seen in the parental BXSB strain, developed in three of these congenic strains. Early onset, severe disease was observed in B10.Yaa.BXSB-Bxs2/3 congenic mice and caused 50% mortality by 12 mo. In B10.Yaa.BXSB-Bxs1/4 mice disease progressed more slowly, resulting in 13% mortality at 12 mo. The progression of renal disease in both of these strains was correlated with the level of anti-dsDNA Abs. B10.Yaa.BXSB-Bxs1 mice, despite their genetic similarity to B10.Yaa.BXSB-Bxs1/4 mice, developed a low-grade glomerulonephritis in the absence of anti-dsDNA Abs. Thus, Bxs4 directed an increase in titer and spectrum of autoantibodies, whereas Bxs1 promoted the development of nephritis. The Bxs2 interval was linked to the production of anti-dsDNA Abs without concomitant glomerulonephritis. In contrast, the Bxs3 interval was sufficient to generate classic lupus nephritis in a nonautoimmune–prone strain. Immune phenotype differed between controls and congenics with a significant increase in B220+ cells in BXSB and B10.Yaa.BXSB-Bxs2/3, and an increase in CD4 to CD8 ratio in both BXSB and B10.Yaa.BXSB-Bxs1/4. Disease in the Bxs3 mice was delayed in comparison to the BXSB parental strain, emphasizing the necessity for multiple interactions in the production of the full BXSB phenotype.
Early Divergence of Fcε Receptor I Signals for Receptor Up-Regulation and Internalization from Degranulation, Cytokine Production, and Survival
Authors: Kitaura Jiro , Xiao Wenbin , Maeda-Yamamoto Mari , Kawakami Yuko , Lowell Clifford A. , Kawakami Toshiaki
Published: April 2014

Mast cells play a critical role in IgE-dependent immediate hypersensitivity. Monomeric IgE binding to its high affinity receptor (FcεRI) results in a number of biological outcomes in mouse mast cells, including increased surface expression of FcεRI and enhanced survival. IgE molecules display heterogeneity in inducing cytokine production; highly cytokinergic IgEs cause extensive FcεRI aggregation, leading to potent enhancement of survival and other activation events, whereas poorly cytokinergic IgEs can do so less efficiently. In this study, we demonstrate that IgE-induced receptor up-regulation is not sensitive to monovalent hapten, which can prevent receptor aggregation induced by IgE, whereas other activation events such as receptor internalization, degranulation, IL-6 production, and survival are sensitive to monovalent hapten. IgE-induced receptor up-regulation is also unique in that no Src family kinases, Syk, or Btk are required for it. By contrast, highly cytokinergic IgE-induced receptor internalization is dependent on Lyn, but not other Src family kinases, Syk, or Btk, whereas degranulation, IL-6 production, and survival require Syk. Weak to moderate stimulation with IgE plus anti-IgE or IgE plus Ag enhances survival, while stronger signals are required for degranulation and IL-6 production. Collectively, signals emanated from IgE-bound FcεRI for receptor up-regulation and internalization are shown to diverge at the receptor or receptor-proximal levels from those for other biological outcomes.
Induction of a CD4+ T Regulatory Type 1 Response by Cyclooxygenase-2-Overexpressing Glioma
Authors: Akasaki Yasuharu , Liu Gentao , Chung Nancy H. C. , Ehtesham Moneeb , Black Keith L. , Yu John S.
Published: April 2014

PGE2, synthesized by cyclooxygenase-2 (COX-2)-overexpressing tumor, is known to contribute to cellular immune suppression in cancer patients, but the mechanism remains unclear. We report the mechanism of a CD4+ T regulatory type 1 (Tr1) induction by CD11c+ mature dendritic cells (DCs) that phagocytose allogeneic and autologous COX-2-overexpressing glioma. A human glioma cell line, U-87MG, and primary cultured glioblastoma cells (MG-377) overexpressed COX-2. We did not detect IL-10Rα expression in these gliomas, and rIL-10 did not suppress their COX-2 expression. Exposure to COX-2-overexpressing glioma induced mature DCs to overexpress IL-10 and decreased IL-12p70 production. These DCs induced a Tr1 response, which is characterized by robust secretion of IL-10 and TGF-β with negligible IL-4 secretion by CD4+ T cells, and an inhibitory effect on admixed lymphocytes. Peripheral CD4+ T cell populations isolated from an MG-377 patient also predominantly demonstrated a Tr1 response against MG-377 cells. Selective COX-2 inhibition in COX-2-overexpressing gliomas at the time of phagocytic uptake by DCs abrogated this regulatory response and instead elicited Th1 activity. COX-2 stable transfectants in LN-18 (LN-18-COX2) also induced a Tr1 response. The effect of a COX-2 inhibition in LN-18-COX2 is reversible after administration of PGE2. Taken together, robust levels of PGE2 from COX-2-overexpressing glioma, which is unresponsive to IL-10 within the local microenvironment, may cause DCs to secrete high levels of IL-10. These results indicate that COX-2-overexpressing tumors induce a Tr1 response, which is mediated by tumor-exposed, IL-10-enhanced DCs.
Repertoire and Immunofocusing of CD8 T Cell Responses Generated by HIV-1 gag-pol and Expression Library Immunization Vaccines
Authors: Singh Rana A. K. , Barry Michael A.
Published: April 2014

Several gene-based vaccine approaches are being tested to drive multivalent cellular immune responses to control HIV-1 viral variants. To compare the utility of these approaches, HLA-A*0201 transgenic mice were genetically immunized with plasmids encoding wild-type (wt) gag-pol, codon-optimized (CO) gag-pol, and an expression library immunization (ELI) vaccine genetically re-engineered to express non-CO fragments of gag and pol fused to ubiquitin for proteasome targeting. Equimolar delivery of each vaccine into HLA-A*0201 transgenic mice generated CD8 T cell responses, with the ELI vaccine producing up to 10-fold higher responses than the wt or CO gag-pol plasmids against cognate and mutant epitopes. All three vaccines generated multivalent CD8 responses against varying numbers of epitopes after priming. However, when the animals were immunized again, the wt and CO gag-pol vaccines boosted only the responses against a subset of epitopes and attenuated the responses against all other Ags including epitopes from clade and drug-resistant viral variants. In contrast, the ELI vaccine boosted CD8 responses against all of the gag-pol Ags and against mutant epitopes from clade and drug-resistant variants. These data suggest that HIV-1 vaccines expressing structurally intact gag and pol proteins drive immunofocused CD8 responses that reduce the repertoire of T cell responses. In contrast, the genetically re-engineered ELI vaccine appears to better maintain the multivalent CD8 responses that may be required to control HIV-1 viral variants.