bluuebunny/crossref_metadata_2025 huggingface dataset:
Human CD1D Gene Has TATA Boxless Dual Promoters: An SP1-Binding Element Determines the Function of the Proximal Promoter
Authors: Chen Qiao-Yi , Jackson Natalie
Published: April 2014

CD1d presents lipid Ags to a specific population of NK T cells, which are involved in the host immune defense, suppression of autoimmunity, and the rejection of tumor cells. The transcriptional control mechanism that determines the regulation and the tissue distribution of CD1d remains largely unknown. After investigating 3.7 kb 5′ upstream of the coding region, we found that human gene encoding CD1d molecule (CD1D) has TATA boxless dual promoters with multiple transcription initiation sites. The proximal promoter is located within the region of −106 to +24, and the distal promoter is located within the region of −665 to −202 with the A of the translational start codon defined as +1. The longest 5′-untranslated region derived from 5′-RACE and apparently generated by the distal promoter has 272 bp in length covering the genomic sequence of the proximal promoter. The region covering the proximal promoter gave a much higher luciferase activity in Jurkat cells than in K562 cells, whereas it was in reverse for the region covering the distal promoter, indicating a cell type sp. act. of the two promoters. Transcription factor SP1 plays a crucial role in the function of the proximal promoter. The analysis of the CD1D promoter region indicates that IFN-γ, NF-IL-6, and T cell factor 1/lymphoid enhancer-binding factor 1 are most likely involved in the regulation of CD1d expression. The illustration of the dual CD1D gene promoters will help to reveal the regulatory factors that control CD1d expression and its tissue distribution for a better understanding of the cross-regulation between CD1d and NK T cells.
Reciprocal Control of Forkhead box O 3a and c-Myc via the Phosphatidylinositol 3-Kinase Pathway Coordinately Regulates p27Kip1 Levels
Authors: Chandramohan Vidyalakshmi , Jeay Sébastien , Pianetti Stefania , Sonenshein Gail E.
Published: April 2014

B cell receptor (BCR) engagement of murine WEHI 231 immature B lymphoma cells leads sequentially to a drop in NF-κB and c-Myc, and induction of the p27Kip1 cyclin-dependent kinase inhibitor, which promotes growth arrest and apoptosis. BCR engagement was recently shown to induce a drop in phosphatidylinositol 3-kinase (PI3K)/Akt signaling, preceding the increase in p27. As induction of p27 is due to an increase in gene transcription, we investigated the role of the Forkhead box O (FOXO) transcription factor family, which has been shown to potently induce p27 promoter activity. We demonstrate that pharmacologic inhibitors of PI3K or BCR engagement lead to decreased inactive cytoplasmic levels and increased active functional nuclear FOXO3a. In contrast, inhibition of PI3K/Akt signaling decreased the levels of NF-κB and c-Myc, which has been shown to repress p27 promoter activity. To test the effects of ectopic c-Myc on endogenous p27 levels, WEHI 231 cells stably expressing c-Myc or empty vector DNA were prepared. Ectopic c-Myc blocked the induction of p27 expression upon either inhibition of PI3K or BCR engagement. Thus, p27Kip1 is coordinately regulated via two arms of a signaling pathway that are inversely controlled upon inhibition of PI3K: induction of the activator FOXO3a and down-regulation of the repressor c-Myc.
The Mouse Homologue of the Leukocyte-Associated Ig-Like Receptor-1 Is an Inhibitory Receptor That Recruits Src Homology Region 2-Containing Protein Tyrosine Phosphatase (SHP)-2, but Not SHP-1
Authors: Lebbink Robert Jan , de Ruiter Talitha , Verbrugge Annelies , Bril Wendy S. , Meyaard Linde
Published: April 2014

We report the molecular cloning and characterization of the first leukocyte-associated Ig-like receptor 1 (LAIR-1) homologue in mice that we have named mouse LAIR-1 (mLAIR-1). The mLAIR-1 gene maps to the proximal end of mouse chromosome 7 in a region syntenic with human chromosome 19q13.4 where the leukocyte receptor cluster is located. The protein shares 40% sequence identity with human LAIR-1, has a single Ig-like domain, and contains two immunoreceptor tyrosine-based inhibitory motif-like structures in its cytoplasmic tail. Mouse LAIR-1 is broadly expressed on various immune cells, and cross-linking of the molecule on stably transfected RBL-2H3 and YT.2C2 cells results in strong inhibition of their degranulation and cytotoxic activities, respectively. Upon pervanadate stimulation, the mLAIR-1 cytoplasmic tail becomes phosphorylated, thereby recruiting Src homology region 2-containing tyrosine phosphatase-2. Interestingly, unlike human LAIR-1, Src homology region 2-containing tyrosine phosphatase-1 is not recruited to the mLAIR-1 cytoplasmic tail. Screening human and mouse cell lines for mLAIR-1 and human LAIR-1 binding partners identified several lines expressing putative ligand(s) for both receptors.
Allelic Exclusion at the TCRδ Locus and Commitment to γδ Lineage: Different Modalities Apply to Distinct Human γδ Subsets
Authors: Couedel Chrystelle , Lippert Eric , Bernardeau Karine , Bonneville Marc , Davodeau François
Published: April 2014

Expression of a β-chain, as a pre-TCR, in T cell precursors prevents further rearrangements on the alternate β allele through a strict allelic exclusion process and enables precursors to undergo differentiation. However, whether allelic exclusion applies to the TCRδ locus is unknown and the role of the γδ TCR in γδ lineage commitment is still unclear. Through the analysis of the rearrangement status of the TCRγ, δ, and β loci in human γδ T cell clones, expressing either the TCR Vδ1 or Vδ2 variable regions, we show that the rate of partial rearrangements at the δ locus is consistent with an allelic exclusion process. The overrepresentation of clones with two functional TCRγ chains indicates that a γδ TCR selection process is required for the commitment of T cell precursors to the γδ lineage. Finally, while complete TCRβ rearrangements were observed in several Vδ2 T cell clones, these were seldom found in Vδ1 cells. This suggests a competitive αβ/γδ lineage commitment in the former subset and a precommitment to the γδ lineage in the latter. We propose that these distinct behaviors are related to the developmental stage at which rearrangements occur, as suggested by the patterns of accessibility to recombination sites that characterize the Vδ1 and Vδ2 subsets.
Induction in Humans of CD8+ and CD4+ T Cell and Antibody Responses by Sequential Immunization with Malaria DNA and Recombinant Protein
Authors: Wang Ruobing , Epstein Judith , Charoenvit Yupin , Baraceros Fe Maria , Rahardjo Nancy , Gay Tanya , Banania Jo-Glenna , Chattopadhyay Rana , de la Vega Patricia , Richie Thomas L. , Tornieporth Nadia , Doolan Denise L. , Kester Kent E. , Heppner D. Gray , Norman Jon , Carucci Daniel J. , Cohen Joe D. , Hoffman Stephen L.
Published: April 2014

Vaccine-induced protection against diseases like malaria, AIDS, and cancer may require induction of Ag-specific CD8+ and CD4+ T cell and Ab responses in the same individual. In humans, a recombinant Plasmodium falciparum circumsporozoite protein (PfCSP) candidate vaccine, RTS,S/adjuvant system number 2A (AS02A), induces T cells and Abs, but no measurable CD8+ T cells by CTL or short-term (ex vivo) IFN-γ ELISPOT assays, and partial short-term protection. P. falciparum DNA vaccines elicit CD8+ T cells by these assays, but no protection. We report that sequential immunization with a PfCSP DNA vaccine and RTS,S/AS02A induced PfCSP-specific Abs and Th1 CD4+ T cells, and CD8+ cytotoxic and Tc1 T cells. Depending upon the immunization regime, CD4+ T cells were involved in both the induction and production phases of PfCSP-specific IFN-γ responses, whereas, CD8+ T cells were involved only in the production phase. IFN-γ mRNA up-regulation was detected in both CD45RA− (CD45RO+) and CD45RA+CD4+ and CD8+ T cell populations after stimulation with PfCSP peptides. This finding suggests CD45RA+ cells function as effector T cells. The induction in humans of the three primary Ag-specific adaptive immune responses establishes a strategy for developing immunization regimens against diseases in desperate need of vaccines.
Inhibition of 19-kDa C-Terminal Region of Merozoite Surface Protein-1-Specific Antibody Responses in Neonatal Pups by Maternally Derived 19-kDa C-Terminal Region of Merozoite Surface Protein-1-Specific Antibodies but Not Whole Parasite-Specific Antibodies
Authors: Stanisic Danielle I. , Martin Laura B. , Gatton Michelle L. , Good Michael F.
Published: April 2014

Immunizing pregnant women with a malaria vaccine is one approach to protecting the mother and her offspring from malaria infection. However, specific maternal Abs generated in response to vaccination and transferred to the fetus may interfere with the infant’s ability to respond to the same vaccine. Using a murine model of malaria, we examined the effect of maternal 19-kDa C-terminal region of merozoite surface protein-1 (MSP119) and Plasmodium yoelii Abs on the pups’ ability to respond to immunization with MSP119. Maternal MSP119-specific Abs but not P. yoelii-specific Abs inhibited Ab production following MSP119 immunization in 2-wk-old pups. This inhibition was correlated with the amount of maternal MSP119 Ab present in the pup at the time of immunization and was due to fewer specific B cells. Passively acquired Ab most likely inhibited the development of an Ab response by blocking access to critical B cell epitopes. If a neonate’s ability to respond to MSP119 vaccination depends on the level of maternal Abs present at the time of vaccination, it may be necessary to delay immunization until Abs specific for the vaccinating Ag have decreased.
Influenza A Vaccine Based on the Extracellular Domain of M2: Weak Protection Mediated via Antibody-Dependent NK Cell Activity
Authors: Jegerlehner Andrea , Schmitz Nicole , Storni Tazio , Bachmann Martin F.
Published: April 2014

Vaccination of mice with a peptide corresponding to the extracellular part of M2 protein coupled to the immunodominant domain of hepatitis B core can protect mice from a lethal challenge with influenza A virus. As the extracellular part of M2 protein is highly conserved in all known human influenza A strains, such a vaccine may protect against all human influenza A strains, which would represent a major advantage over current vaccine strategies. The present study demonstrates that protection is mediated exclusively by Abs, a very important feature of a successful preventive vaccine. However, these Abs neither bind efficiently to the free virus nor neutralize virus infection, but bind to M2 protein expressed on the surface of virus-infected cells. The presence of NK cells is important for protection, whereas complement is not, supposing that protection is mediated via Ab-dependent, cell-mediated cytotoxicity. The absence of neutralizing Abs results in much weaker protection than that achieved by vaccination with UV-inactivated influenza virus. Specifically, whereas neutralizing Abs completely eliminate signs of disease even at high viral challenge doses, M2-specific Abs cannot prevent infection, but merely reduce disease at low challenge doses. M2-specific Abs fail to protect from high challenge doses, as vaccinated mice undergo lethal infection under these conditions. In conclusion, protection mediated by M2-hepatitis B core vaccine would be insufficient during the yearly epidemics, for which full protection is desirable, and overall is clearly inferior to protection achieved by immunization with classical inactivated viral preparations.
Distinctive Roles for 2′,5′-Oligoadenylate Synthetases and Double-Stranded RNA-Dependent Protein Kinase R in the In Vivo Antiviral Effect of an Adenoviral Vector Expressing Murine IFN-β
Authors: Al-khatib Khaldun , Williams Bryan R. G. , Silverman Robert H. , Halford William , Carr Daniel J. J.
Published: April 2014

To evaluate the anti-HSV-1 mechanisms of murine IFN-β in ocular infection, mice were transduced with an adenoviral vector expressing murine IFN-β (Ad:IFN-β). Ocular transduction with Ad:IFN-β resulted in enhanced survival following infection with HSV-1. The protective effect was associated with a reduction in 1) viral titer, 2) viral gene expression, 3) IFN-γ levels, and 4) the percentage of CD8+ T lymphocyte and NK cell infiltration in infected tissue. Expression of IFN-β resulted in an elevation of the IFN-induced antiviral gene 2′,5′-oligoadenylate synthetase (OAS1a) but not dsRNA-dependent protein kinase R (PKR) in the cornea and trigeminal ganglion (TG). Mice deficient in the downstream effector molecule of the OAS pathway, RNase L, were no more sensitive to ocular HSV-1 compared with wild-type controls in the TG based on measurements of viral titer. However, the efficacy of Ad:IFN-β was transiently lost in the eyes of RNase L mice. By comparison, PKR-deficient mice were more susceptible to ocular HSV-1 infection, and the antiviral efficacy following transduction with Ad:IFN-β was significantly diminished in the eye and TG. These results suggest that PKR is central in controlling ocular HSV-1 infection in the absence of exogenous IFN, whereas the OAS pathway appears to respond to exogenous IFN, contributing to the establishment of an antiviral environment in a tissue-restricted manner.
IL-6 Prevents T Cell-Mediated Hepatitis via Inhibition of NKT Cells in CD4+ T Cell- and STAT3-Dependent Manners
Authors: Sun Rui , Tian Zhigang , Kulkarni Shalini , Gao Bin
Published: April 2014

The hepatoprotective effect of IL-6 on various forms of liver injury including T cell-mediated hepatitis has been well documented, and it is believed that induction of antiapoptotic proteins is an important mechanism. In this study, we provide evidence suggesting an additional mechanism involved in the protective role of IL-6 in T cell-mediated hepatitis. In NKT cell-depleted mice, Con A-induced liver injury is diminished; this can be restored by the adoptive transfer of liver mononuclear cells or NKT cells from wild-type mice, but not from IL-6-treated mice. In vitro IL-6 treatment inhibits the ability of mononuclear cells to restore Con A-induced liver injury in NKT-depleted mice, whereas the same treatment does not inhibit purified NKT cells from restoring the injury. The addition of CD3+ T cells or CD4+ T cells can restore the inhibitory effect of IL-6 on purified NKT cells, whereas the addition of CD3+ T cells from CD4-deficient mice fails to restore this inhibitory effect. The expression of IL-6R was detected in 52.6% of hepatic CD3+ T cells and 32.7% of hepatic CD4+ T cells, but only in 3.9% of hepatic NK and 1.5% of hepatic NKT cells. Finally, treatment with IL-6 induces STAT3 activation in hepatic lymphocytes and hepatic T cells, and blocking such activation abolishes the inhibitory effect of IL-6 on hepatic lymphocytes to restore liver injury. Taken together, these findings suggest that in addition to its antiapoptotic abilities, as previously well documented, IL-6/STAT3 inhibits NKT cells via targeting CD4+ T cells and consequently prevents T cell-mediated hepatitis.
Arthritogenic Properties of Double-Stranded (Viral) RNA
Authors: Zare Fariba , Bokarewa Maria , Nenonen Nancy , Bergström Thomas , Alexopoulou Lena , Flavell Richard A. , Tarkowski Andrej
Published: April 2014

Viral infections often lead to arthralgias and overt arthritic states. The inflammatogenic compound of the viruses giving rise to such an outcome has to date not been identified. Because expression of dsRNA is a common feature of all viruses, we decided to analyze whether this property leads to the induction of arthritis. Histological signs of arthritis were evident already on day 3 following intra-articular administration of dsRNA. Arthritis was characterized by infiltration of macrophages into synovial tissue. It was not dependent on acquired immune responses because SCID mice also raised joint inflammation. NF-κB was activated upon in vitro exposure to dsRNA, indicating its role in the induction/progression of arthritis. Importantly, we found that dsRNA arthritis was triggered through IL-1R signaling because mice being deficient for this molecule were unable to develop joint inflammation. Although dsRNA is typically recognized by Toll-like receptor 3, Toll-like receptor 3 knockout mice developed arthritis, indicating that some other receptors are instrumental in the inducing of inflammation. Our results from in vitro experiments indicate that proinflammatory cytokines and chemokines stimulating monocyte influx were readily triggered in response to stimulation with dsRNA. These findings demonstrate that viral dsRNA is clearly arthritogenic. Importantly, macrophages and their products play an important role in the development of arthritis triggered by dsRNA.
Immunopathogenesis of Experimental Ulcerative Colitis Is Mediated by Eosinophil Peroxidase
Authors: Forbes Elizabeth , Murase Tosei , Yang Ming , Matthaei Klaus I. , Lee James J. , Lee Nancy A. , Foster Paul S. , Hogan Simon P.
Published: April 2014

The precise role that individual inflammatory cells and mediators play in the development of gastrointestinal (GI) dysfunction and extraintestinal clinical manifestations of ulcerative colitis (UC) is unknown. In this study, we have used a mouse model of UC to establish a central role for eotaxin and, in turn, eosinophils in the development of the immunopathogenesis of this disease. In this model the administration of dextran sodium sulfate (DSS) induces a prominent colonic eosinophilic inflammation and GI dysfunction (diarrhea with blood and shortening of the colon) that resembles UC in patients. GI dysfunction was associated with evidence of eosinophilic cytolytic degranulation and the release of eosinophil peroxidase (EPO) into the colon lumen. By using IL-5 or eotaxin-deficient mice, we show an important role for eotaxin in eosinophil recruitment into the colon during experimental UC. Furthermore, using EPO-deficient mice and an EPO inhibitor resorcinol we demonstrate that eosinophil-derived peroxidase is critical in the development of GI dysfunction in experimental UC. These findings provide direct evidence of a central role for eosinophils and EPO in GI dysfunction and potentially the immunopathogenesis of UC.
Endothelial Induction of fgl2 Contributes to Thrombosis during Acute Vascular Xenograft Rejection
Authors: Ghanekar Anand , Mendicino Michael , Liu Hao , He Wei , Liu Mingfeng , Zhong Robert , Phillips M. James , Levy Gary A. , Grant David R.
Published: April 2014

Thrombosis is a prominent feature of acute vascular rejection (AVR), the current barrier to survival of pig-to-primate xenografts. Fibrinogen-like protein 2 (fgl2/fibroleukin) is an inducible prothrombinase that plays an important role in the pathogenesis of fibrin deposition during viral hepatitis and cytokine-induced fetal loss. We hypothesized that induction of fgl2 on the vascular endothelium of xenografts contributes to thrombosis associated with AVR. We first examined fgl2 as a source of procoagulant activity in the pig-to-primate combination. The porcine fgl2 (pfgl2) was cloned and its chromosomal locus was identified. Recombinant pfgl2 protein expressed in vitro was detected on the cell surface and generated thrombin from human prothrombin. Studies of pig-to-baboon kidney xenografts undergoing AVR in vivo revealed induction of pfgl2 expression on graft vascular endothelial cells (ECs). Cultured porcine ECs activated by human TNF-α in vitro demonstrated induction of pfgl2 expression and enhanced activation of human prothrombin. The availability of gene-targeted fgl2-deficient mice allowed the contribution of fgl2 to the pathogenesis of AVR to be directly examined in vivo. Hearts heterotopically transplanted from fgl2+/+ and fgl2+/− mice into Lewis rats developed AVR with intravascular thrombosis associated with induction of fgl2 in graft vascular ECs. In contrast, xenografts from fgl2−/− mice were devoid of thrombosis. These observations collectively suggest that induction of fgl2 on the vascular endothelium plays a role in the pathogenesis of AVR-associated thrombosis. Manipulation of fgl2, in combination with other interventions, may yield novel strategies by which to overcome AVR and extend xenograft survival.
Effects of Complement C5 on Apoptosis in Experimental Autoimmune Encephalomyelitis
Authors: Niculescu Teodora , Weerth Susanna , Niculescu Florin , Cudrici Cornelia , Rus Violeta , Raine Cedric S. , Shin Moon L. , Rus Horea
Published: April 2014

Complement activation is involved in the initiation of Ab-mediated inflammatory demyelination in experimental autoimmune encephalomyelitis (EAE). At a sublytic dose, the C5b-9 membrane attack complex protects oligodendrocytes (OLG) from apoptosis. Using C5-deficient (C5-d) mice, we previously showed a dual role for C5: enhancement of inflammatory demyelination in acute EAE, and promotion of remyelination during recovery. In this study, we investigated the role of C5 in apoptosis in myelin-induced EAE. In acute EAE, C5-d and C5-sufficient (C5-s) mice had similar numbers of total apoptotic cells, whereas C5-s had significantly fewer than C5-d during recovery. In addition, although both groups of mice displayed TUNEL+ OLG, there were significantly fewer in C5-s than in C5-d during both acute EAE and recovery. Gene array and immunostaining of apoptosis-related genes showed that Fas ligand expression was higher in C5-s. In C5-s mice, Fas+ cells were also higher than in C5-d mice in acute EAE; however, these cells were significantly reduced during recovery. Together, these findings are consistent with the role of C5, possibly by forming the membrane attack complex, in limiting OLG apoptosis in EAE, thus promoting remyelination during recovery.
Substance P Augments Borrelia burgdorferi-Induced Prostaglandin E2 Production by Murine Microglia
Authors: Rasley Amy , Marriott Ian , Halberstadt Craig R. , Bost Kenneth L. , Anguita Juan
Published: April 2014

Substance P is a ubiquitous CNS neuropeptide and has recently been demonstrated to augment immune cell function during inflammatory events. Central to the ability of substance P to modulate immune cell function is the interaction of substance P with the substance P neurokinin-1 receptor expressed by a variety of immune cells, including microglia. CNS involvement during Lyme disease can occur when Borrelia burgdorferi, the causative agent of Lyme disease, gains access to the CNS. In the present study, we demonstrate that substance P augments B. burgdorferi-induced expression of mRNA encoding COX-2 and subsequent secretion of PGE2 by cultured, murine microglia. Furthermore, this effect is associated with the ability of substance P to enhance B. burgdorferi-induced NF-κB activation, as demonstrated by increased nuclear localization of the p65 (RelA) subunit of NF-κB in these cells. Interestingly, we demonstrate that substance P augments B. burgdorferi-induced expression of mRNA encoding two PGE2 receptors, E-prostanoid receptor subtypes 2 and 4, as well as each receptor protein. In addition, these effects are mediated via interactions between substance P and its high affinity receptor, as evidenced by the absence of augmented PGE2 synthesis in the presence of a specific neurokinin-1 receptor antagonist or in cells genetically deficient in the expression of these receptors. Taken together, the present demonstration that substance P can exacerbate B. burgdorferi-induced inflammatory responses in microglia in vitro may indicate a role for this neuropeptide in the development of CNS inflammation observed during human neuroborreliosis.
Phosphoinositide 3-Kinase and Akt Occupy Central Roles in Inflammatory Responses of Toll-Like Receptor 2-Stimulated Neutrophils
Authors: Strassheim Derek , Asehnoune Karim , Park Jong-Sung , Kim Jae-Yeol , He Qianbin , Richter Donald , Kuhn Katherine , Mitra Sanchayita , Abraham Edward
Published: April 2014

Neutrophils are critical initiators and effectors of the innate immune system and express Toll-like receptor 2 (TLR2) and TLR4. Although signaling through pathways involving phosphoinositide 3-kinase (PI3-K) and the downstream kinase Akt (protein kinase B) plays a central role in modulating neutrophil chemotaxis and superoxide generation in response to engagement of G protein-coupled receptors, the importance of these kinases in affecting inflammatory responses of neutrophils stimulated through TLR2 has not been examined. In these experiments, we found activation of Akt in neutrophils stimulated with the TLR2-specific ligands peptidoglycan and the lipopeptide tri-palmitoyl-S-glyceryl-Cys-Ser-(Lys)4 that occurred earlier and was of greater magnitude than that present after exposure to the TLR4 agonist LPS. The release of the proinflammatory mediators TNF-α and macrophage inflammatory protein-2 was inhibited in a dose-dependent manner by PI3-K blockade. The IC50 for inhibition of peptidoglycan-stimulated Akt activation and macrophage inflammatory protein-2 release correlated closely, indicating linkage of these two events. PI3-K blockade did not inhibit nuclear translocation of NF-κB, but did prevent Ser536 phosphorylation of the p65 subunit of NF-κB, an event required for maximal transcriptional activity of NF-κB. Inhibition of PI3-K also prevented activation of p38 mitogen-activated protein kinase and extracellular receptor-activated kinase 1/2 in TLR2-stimulated neutrophils. These results demonstrate that the PI3-K-Akt axis occupies a central role in TLR2-induced activation of neutrophils.
Regulation of Postsurgical Fibrosis by the Programmed Death-1 Inhibitory Pathway
Authors: Holsti Matthew A. , Chitnis Tanuja , Panzo Ronald J. , Bronson Roderick T. , Yagita Hideo , Sayegh Mohamed H. , Tzianabos Arthur O.
Published: April 2014

Surgical adhesions are a common and often severe complication of abdominal or pelvic injury that cause pelvic pain, bowel obstruction, and infertility in women. Current treatments are of limited effectiveness because little is known about the cellular and subcellular processes underlying adhesiogenesis. Recently, we showed that Th1 αβ CD4+ T cells mediate the pathogenesis of adhesion formation in a rodent model of this disease process. In this study, we demonstrate that in mice these T cells home directly to the site of surgically induced adhesions and control local chemokine production in a manner dependent on the CD28 T cell costimulatory pathway. Conversely, the inhibitory programmed death-1 pathway plays a central role in limiting adhesiogenesis, as programmed death-1 blockade was associated with increased T cell infiltration, chemokine production, and a concomitant exacerbation of disease. Our results reveal for the first time that the development of postsurgical fibrosis is under the tight control of positive and negative T cell costimulation, and suggest that targeting these pathways may provide promising therapies for the prevention of adhesion formation.
Lymphomagenesis, Hydronephrosis, and Autoantibodies Result from Dysregulation of IL-9 and Are Differentially Dependent on Th2 Cytokines
Authors: Lauder Angus J. , Jolin Helen E. , Smith Philippa , van den Berg José G. , Jones Alison , Wisden William , Smith Kenneth G. C. , Dasvarma Ayan , Fallon Padraic G. , McKenzie Andrew N. J.
Published: April 2014

Interleukin-9 is an immunoregulatory cytokine implicated in the development of asthma and allergy. To investigate the role of IL-9 in vivo, we have generated transgenic mice in which IL-9 is expressed from its own promoter. Strikingly, overexpression of IL-9 resulted in premature mortality associated with a complex phenotype characterized by the development of autoantibodies, hydronephrosis, and T cell lymphoma. By intercrossing IL-9 transgenic mice with a panel of Th2 cytokine-deficient mice, we demonstrate that these disorders represent distinct phenotypes that can be dissociated by their differential dependence on Th2 cytokines. Autoantibody production was ablated in IL-9 transgenic animals with a combined absence of IL-4, IL-5, and IL-13, coincident with a reduction in peritoneal B-1 cells. Hydronephrosis arose in 75% of IL-9 transgenic animals and was dependent on the presence of IL-4 and IL-13. In contrast, T cell lymphomas developed independently of the other Th2 cytokines, with the generation of rapidly proliferating CD8+ or CD4+CD8+ T cell clones that arose in the thymus before infiltrating both lymphoid and nonlymphoid tissues. Our data highlight potentially important new roles for IL-9, through its regulation of downstream Th2 effector cytokines, in autoantibody production and in hydronephrosis.
B Cell Positive Selection: Road Map to the Primary Repertoire?
Authors: Cancro Michael P. , Kearney John F.
Published: April 2014

Accumulating evidence indicates that positive selection events mediate differentiation, lineage commitment, and longevity of B lymphocytes. The BCR plays a central role, dictating the likelihood that newly formed cells will complete maturation, as well as whether cells persist within mature pools. Competition among B cells for limited, life span-promoting resources, which include self-ligands, lineage-specific cytokines, and innate receptor ligands, underlie these selective processes. Together, these observations suggest that positive selection is a critical feature in the establishment and maintenance of all lymphocyte pools, prompting re-evaluation of the underlying biological rationale for this process.
The Diabetes Susceptibility Locus Idd5.1 on Mouse Chromosome 1 Regulates ICOS Expression and Modulates Murine Experimental Autoimmune Encephalomyelitis
Authors: Greve Bernhard , Vijayakrishnan Lalitha , Kubal Aarup , Sobel Raymond A. , Peterson Laurence B. , Wicker Linda S. , Kuchroo Vijay K.
Published: April 2014

Linkage analysis and congenic mapping in NOD mice have identified a susceptibility locus for type 1 diabetes, Idd5.1 on mouse chromosome 1, which includes the Ctla4 and Icos genes. Besides type 1 diabetes, numerous autoimmune diseases have been mapped to a syntenic region on human chromosome 2q33. In this study we determined how the costimulatory molecules encoded by these genes contribute to the immunopathogenesis of experimental autoimmune encephalomyelitis (EAE). When we compared levels of expression of costimulatory molecules on T cells, we found higher ICOS and lower full-length CTLA-4 expression on activated NOD T cells compared with C57BL/6 (B6) and C57BL/10 (B10) T cells. Using NOD.B10 Idd5 congenic strains, we determined that a 2.1-Mb region controls the observed expression differences of ICOS. Although Idd5.1 congenic mice are resistant to diabetes, we found them more susceptible to myelin oligodendrocyte glycoprotein 35–55-induced EAE compared with NOD mice. Our data demonstrate that higher ICOS expression correlates with more IL-10 production by NOD-derived T cells, and this may be responsible for the less severe EAE in NOD mice compared with Idd5.1 congenic mice. Paradoxically, alleles at the Idd5.1 locus have opposite effects on two autoimmune diseases, diabetes and EAE. This may reflect differential roles for costimulatory pathways in inducing autoimmune responses depending upon the origin (tissue) of the target Ag.
Fine Mapping, Gene Content, Comparative Sequencing, and Expression Analyses SupportCtla4andNramp1as Candidates forIdd5.1andIdd5.2in the Nonobese Diabetic Mouse
Authors: Wicker Linda S. , Chamberlain Giselle , Hunter Kara , Rainbow Dan , Howlett Sarah , Tiffen Paul , Clark Jan , Gonzalez-Munoz Andrea , Cumiskey Anne Marie , Rosa Raymond L. , Howson Joanna M. , Smink Luc J. , Kingsnorth Amanda , Lyons Paul A. , Gregory Simon , Rogers Jane , Todd John A. , Peterson Laurence B.
Published: April 2014

At least two loci that determine susceptibility to type 1 diabetes in the NOD mouse have been mapped to chromosome 1, Idd5.1 (insulin-dependent diabetes 5.1) and Idd5.2. In this study, using a series of novel NOD.B10 congenic strains, Idd5.1 has been defined to a 2.1-Mb region containing only four genes, Ctla4, Icos, Als2cr19, and Nrp2 (neuropilin-2), thereby excluding a major candidate gene, Cd28. Genomic sequence comparison of the two functional candidate genes, Ctla4 and Icos, from the B6 (resistant at Idd5.1) and the NOD (susceptible at Idd5.1) strains revealed 62 single nucleotide polymorphisms (SNPs), only two of which were in coding regions. One of these coding SNPs, base 77 of Ctla4 exon 2, is a synonymous SNP and has been correlated previously with type 1 diabetes susceptibility and differential expression of a CTLA-4 isoform. Additional expression studies in this work support the hypothesis that this SNP in exon 2 is the genetic variation causing the biological effects of Idd5.1. Analysis of additional congenic strains has also localized Idd5.2 to a small region (1.52 Mb) of chromosome 1, but in contrast to the Idd5.1 interval, Idd5.2 contains at least 45 genes. Notably, the Idd5.2 region still includes the functionally polymorphic Nramp1 gene. Future experiments to test the identity of Idd5.1 and Idd5.2 as Ctla4 and Nramp1, respectively, can now be justified using approaches to specifically alter or mimic the candidate causative SNPs.
Mouse Complement Receptor-Related Gene y/p65-Neutralized Tumor Vaccine Induces Antitumor Activity In Vivo
Authors: Ohta Rieko , Kondor Natalie , Dohi Natsuki , Tomlinson Stephen , Imai Masaki , Holers V. Michael , Okada Hidechika , Okada Noriko
Published: April 2014

Two mouse tumor cell lines, Meth A (BALB/c mouse-derived fibrosarcoma) and MM46 (C3H/He mouse-derived mammary tumor), were shown to express high levels of complement receptor-related gene y/p65 (Crry/p65), a membrane-bound complement-regulatory protein. Inhibiting the complement-regulatory activity of Crry/p65 with mAb 5D5 induced high levels of C3 deposition on in vivo tumor-derived Meth A and MM46 cells. To determine the effect of Crry/p65 blockade and increased C3 deposition on in vivo tumor growth, Meth A and MM46 cells were treated with 5D5 mAb and injected into BALB/c and C3H/He mice, respectively. Pretreating MM46 cells with 5D5 mAb significantly suppressed their tumorigenicity when injected s.c. Pretreatment with 5D5 mAb had a modest effect on Meth A s.c. tumor growth. Because complement is involved in the induction of an immune response, we investigated the effect of Crry/p65 blockade and increased C3 deposition on the immunogenicity of the tumor cells in a vaccination protocol. Vaccination of mice with irradiated Meth A cells pretreated with 5D5 mAb protected mice from subsequent challenge. In contrast, vaccination with irradiated Meth A cells without pretreatment was not protective. Survival was correlated with a high titer IgM response and specific CTL activity. These data demonstrate that the functional inhibition of Crry/p65 on tumor cells affects tumor growth and immunogenicity, and that the complement deposition resulting from this inhibition can act in concert with antitumor effector mechanisms to elicit potent antitumor immunity in vivo.
Different Costimulatory and Growth Factor Requirements for CD4+ and CD8+ T Cell-Mediated Rejection
Authors: Vu Minh Diem , Amanullah Farhana , Li Yongsheng , Demirci Gulcin , Sayegh Mohamed H. , Li Xian Chang
Published: April 2014

Costimulatory signals and growth factor signals play a key role in commanding T cell activation and T cell effector function. However, how costimulatory signals and growth factor signals interact and integrate into the activation program of CD4+ and CD8+ T cells during the allograft response remains poorly defined. In the present study we found that either CD4- or CD8-deficient mice can vigorously reject the skin allografts. Blocking rapamycin-sensitive growth factor signals produced long term skin allograft survival in CD4-deficient mice (mean survival time, >120 days), but not in CD8-deficient mice (mean survival time, 20 days). Analysis of CFSE-labeled cells proliferating in the allogeneic hosts revealed that clonal expansion of CD4+ T cells in vivo was more resistant to growth factor blockade than that of CD8+ T cells. However, blockade or genetic absence of CD28/CD154 costimulatory molecules rendered CD4+ T cell-mediated rejection sensitive to rapamycin, and long term skin allograft survival can be readily induced by rapamycin in the absence of CD28/CD154 signals (>100 days). Furthermore, blocking OX40 costimulation induced long term skin allograft survival in CD4-deficient mice and CD8-deficient mice when both CD28 and CD154 were transiently blocked. We conclude that CD4+ and CD8+ T cells exhibit distinct sensitivity to growth factor blockade in transplant rejection, and CD28/CD154-independent rejection is sensitive to rapamycin and appears to be supported by OX40 costimulation.
Antigen Distribution Drives Programmed Antitumor CD8 Cell Migration and Determines Its Efficiency
Authors: Boissonnas Alexandre , Combadiere Christophe , Lavergne Elise , Maho Maud , Blanc Catherine , Debré Patrice , Combadiere Behazine
Published: April 2014

Understanding both the role of tumor Ag in CD8 cell differentiation and the reasons that CD8 cells may work inefficiently is crucial for therapeutic approaches in cancer. We studied OT-1 CD8 cell responses in vivo in a differential Ag-distribution model that used EG-7, the EL-4 thymoma transfected with OVA. On their initial Ag encounter, OT-1 CD8 cells underwent programmed expansion in the lymph nodes, where they acquired the ability to migrate to the encapsulated tumor site after ≥4 divisions, without continuous antigenic stimulation. This short antigenic stimulation was sufficient to induce the migration differentiation program, which included modulation of chemokine receptor mRNA expression and down-regulation of CD62L. Moreover, Ag quantity determined the behavior of the OT-1 CD8 cells, including their effector functions and sensitivity to apoptosis. Thus, the initial Ag encounter drives the programmed cell migration potencies, but neither effector functions nor cell death can occur without continuous TCR triggering.
Delayed and Restricted Expression Limits Putative Instructional Opportunities of Vγ1.1/Vγ2 γδ TCR in αβ/γδ Lineage Choice in the Thymus
Authors: Krotkova Anna , Smith Emma , Nerz Gabi , Falk Ingrid , Eichmann Klaus
Published: April 2014

Development of αβ and γδ T cells depends on productive rearrangement of the appropriate TCR genes and their subsequent expression as proteins. TCRβ and TCRγδ proteins first appear in DN3 and DN4 thymocytes, respectively. So far, it is not clear whether this is due to a delayed expression of TCRγδ proteins or to a more rapid progression to DN4 of thymocytes expressing TCRγδ. The answer to this question bears on the distinction between instructive and stochastic models of αβ/γδ lineage decision. To study this question, we first monitored initial TCR protein expression in wild-type and TCR transgenic mice in reaggregate thymic organ cultures. A TCRβ transgene was expressed in nearly all DN3 and DN4 cells, accelerated DN3 to DN4 transition, and strongly diminished the number of cells that express TCRγδ proteins. In contrast, TCRγδ transgenes were expressed only in a fraction of DN4 cells, did not accelerate DN3 to DN4 transition, and did not reduce the number of DN4 cells expressing TCRβ proteins. The TCRβ transgene partially inhibited endogenous TCRγ rearrangements, whereas the TCRγδ transgenes did not inhibit endogenous TCRβ rearrangements. Second, we analyzed frequencies of productive TCRβ and TCRγδ V(D)J junctions in DN3 and DN4 subsets. Most importantly, frequencies of productive TCRγδ rearrangements (Vδ5, Vγ1.1, and Vγ2) appeared unselected in DN3. The results suggest a late and restricted expression of the corresponding γδTCR, severely limiting their putative instructional opportunities in αβ/γδ divergence.
Structural and Functional Characterization of Complement C4 and C1s-Like Molecules in Teleost Fish: Insights into the Evolution of Classical and Alternative Pathways
Authors: Boshra Hani , Gelman Andrew E. , Sunyer J. Oriol
Published: April 2014

There is growing evidence that certain components of complement systems in lower vertebrates are promiscuous in their modes of activation through the classical or alternative pathways. To better understand the evolution of the classical pathway, we have evaluated the degree of functional diversification of key components of the classical and alternative pathways in rainbow trout, an evolutionarily relevant teleost species. Trout C4 was purified in two distinct forms (C4-1 and C4-2), both exhibiting the presence of a thioester bond at the cDNA and protein levels. C4-1 and C4-2 bound in a similar manner to trout IgM-sensitized sheep erythrocytes in the presence of Ca2+/Mg2+, and both C4 molecules equally restored the classical pathway-mediated hemolytic activity of serum depleted of C3 and C4. Reconstitution of activity was dependent on the presence of both C3-1 and C4-1/C4-2 and on the presence of IgM bound to the sheep erythrocytes. A C1s-like molecule was shown to cleave specifically purified C4-1 and C4-2 into C4b, while failing to cleave trout C3 molecules. The C1s preparation was unable to cleave trout factor B/C2 when added in the presence of C3b or C4b molecules. Our results show a striking conservation of the mode of activation of the classical pathway. We also show that functional interchange between components of the classical and alternative pathway in teleosts is more restricted than was anticipated. These data suggest that functional diversification between the two pathways must have occurred shortly after the gene duplication that gave rise to the earliest classical pathway molecules.
Serine Residues 286, 288, and 293 within the CIITA: A Mechanism for Down-Regulating CIITA Activity through Phosphorylation
Authors: Greer Susanna F. , Harton Jonathan A. , Linhoff Michael W. , Janczak Christin A. , Ting Jenny P.-Y. , Cressman Drew E.
Published: April 2014

CIITA is the primary factor activating the expression of the class II MHC genes necessary for the exogenous pathway of Ag processing and presentation. Strict control of CIITA is necessary to regulate MHC class II gene expression and induction of an immune response. We show in this study that the nuclear localized form of CIITA is a predominantly phosphorylated form of the protein, whereas cytoplasmic CIITA is predominantly unphosphorylated. Novel phosphorylation sites were determined to be located within a region that contains serine residues 286, 288, and 293. Double mutations of these residues increased nuclear CIITA, indicating that these sites are not required for nuclear import. CIITA-bearing mutations of these serine residues significantly increased endogenous MHC class II expression, but did not significantly enhance trans-activation from a MHC class II promoter, indicating that these phosphorylation sites may be important for gene activation from intact chromatin rather than artificial plasmid-based promoters. These data suggest a model for CIITA function in which phosphorylation of these specific sites in CIITA in the nucleus serves to down-regulate CIITA activity.
Generation of Peptide Mimics of the Epitope Recognized by Trastuzumab on the Oncogenic Protein Her-2/neu
Authors: Riemer Angelika B. , Klinger Markus , Wagner Stefan , Bernhaus Astrid , Mazzucchelli Luca , Pehamberger Hubert , Scheiner Otto , Zielinski Christoph C. , Jensen-Jarolim Erika
Published: April 2014

Immunizations with the oncogenic protein Her-2/neu elicit Abs exerting diverse biological effects--depending on epitope specificity, tumor growth may be inhibited or enhanced. Trastuzumab (herceptin) is a growth-inhibitory humanized monoclonal anti-Her-2/neu Ab, currently used for passive immunotherapy in the treatment of breast cancer. However, Ab therapies are expensive and have to be repeatedly administered for long periods of time. In contrast, active immunizations produce ongoing immune responses. Therefore, the study aims to generate peptide mimics of the epitope recognized by trastuzumab for vaccine formulation, ensuring the subsequent induction of tumor growth inhibitory Abs. We used the phage display technique to generate epitope mimics, mimotopes, complementing the screening Ab trastuzumab. Five candidate mimotopes were isolated from a constrained 10 mer library. These peptides were specifically recognized by trastuzumab, and showed distinctive mimicry with Her-2/neu in two experimental setups. Subsequently, immunogenicity of a selected mimotope was examined in BALB/c mice. Immunizations with a synthetic mimotope conjugated to tetanus toxoid resulted in Abs recognizing Her-2/neu in a blotted cell lysate as well as on the SK-BR-3 cell surface. Analogous to trastuzumab, the induced Abs caused internalization of the receptor from the cell surface to endosomal vesicles. These results indicate that the selected mimotopes are suitable for formulation of a breast cancer vaccine because the resulting Abs show similar biological features as trastuzumab.
A Hierarchy of Nuclear Localization Signals Governs the Import of the Regulatory Factor X Complex Subunits and MHC Class II Expression
Authors: Nagarajan Uma M. , Long Alyssa B. , Harreman Michelle T. , Corbett Anita H. , Boss Jeremy M.
Published: April 2014

Comprised of RFX5, RFXAP, and RFX-B/ANK, the regulatory factor X (RFX) complex is an obligate transcription factor required for the expression of MHC class II genes. RFX functions by binding to the conserved X1 box sequence located upstream of all MHC class II genes. Using a mutagenesis scheme and a yeast heterologous reporter system, the mechanism by which the RFX complex is transported into the nucleus was examined. The results have identified specific nuclear localization signals (NLS) in both RFX5 and RFXAP that direct the nuclear translocation and expression of MHC class II genes. Additionally, a nuclear export signal was identified in the N terminus of RFXAP. RFX-B was poorly localized to the nucleus, and no specific NLS was identified. Whereas RFX5 could import an RFXAP NLS mutant into the nucleus, it had no effect on the import of RFX-B. The results suggest that although RFX5 and RFXAP could assemble before nuclear import, RFX-B association with the complex does not take place until after the subunits enter the nucleus. The identification of nuclear import and export sites on RFX molecules provides potential targets to modulate MHC class II expression.
Immature Dendritic Cells (DCs) Use Chemokines and Intercellular Adhesion Molecule (ICAM)-1, But Not DC-Specific ICAM-3-Grabbing Nonintegrin, to Stimulate CD4+ T Cells in the Absence of Exogenous Antigen
Authors: Real Eliana , Kaiser Andrew , Raposo Graça , Amara Ali , Nardin Alessandra , Trautmann Alain , Donnadieu Emmanuel
Published: April 2014

Dendritic cells (DCs) possess a number of unique features that distinguish them from other APCs. One such feature is their ability to trigger Ag-independent responses in T cells. Previous studies have focused on mature DCs, but the prevalence of this phenomenon in the resting-state immature DCs has never been considered. In this study, we show that, in the absence of Ag, human immature DCs trigger multiple responses in autologous primary CD4+ T cells, namely, increased motility, small Ca2+ transients, and up-regulation of CD69. These responses are particularly marked in CD4+ memory T cells. By using several experimental approaches, we found that DC-specific ICAM-3-grabbing nonintegrin plays no role in the induction of T cell responses, whereas ICAM-1/LFA-1 interactions are required. In addition, DC-produced chemokines contribute to the Ag-independent T cell stimulatory ability of DCs, because pertussis toxin-treated T cells exhibit diminished responses to immature DCs. More particularly, CCL17 and CCL22, which are constitutively produced by immature DCs, mediate both T cell polarization and attraction. Thus, immature DCs owe part of their outstanding Ag-independent T cell stimulatory ability to chemokines and ICAM-1, but not DC-specific ICAM-3-grabbing nonintegrin.