bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vivo Persistence of Codominant Human CD8+ T Cell Clonotypes Is Not Limited by Replicative Senescence or Functional Alteration
Authors: Derré Laurent , Bruyninx Marc , Baumgaertner Petra , Devevre Estelle , Corthesy Patricia , Touvrey Cédric , Mahnke Yolanda D. , Pircher Hanspeter , Voelter Verena , Romero Pedro , Speiser Daniel E. , Rufer Nathalie
Published: April 2014

T cell responses to viral epitopes are often composed of a small number of codominant clonotypes. In this study, we show that tumor Ag-specific T cells can behave similarly. In a melanoma patient with a long lasting HLA-A2/NY-ESO-1-specific T cell response, reaching 10% of circulating CD8 T cells, we identified nine codominant clonotypes characterized by individual TCRs. These clonotypes made up almost the entire pool of highly differentiated effector cells, but only a fraction of the small pool of less differentiated “memory” cells, suggesting that the latter serve to maintain effector cells. The different clonotypes displayed full effector function and expressed TCRs with similar functional avidity. Nevertheless, some clonotypes increased, whereas others declined in numbers over the observation period of 6 years. One clonotype disappeared from circulating blood, but without preceding critical telomere shortening. In turn, clonotypes with increasing frequency had accelerated telomere shortening, correlating with strong in vivo proliferation. Interestingly, the final prevalence of the different T cell clonotypes in circulation was anticipated in a metastatic lymph node withdrawn 2 years earlier, suggesting in vivo clonotype selection driven by metastases. Together, these data provide novel insight in long term in vivo persistence of T cell clonotypes associated with continued cell turnover but not replicative senescence or functional alteration.
Signal-Transducing Adaptor Protein-2 Regulates Integrin-Mediated T Cell Adhesion through Protein Degradation of Focal Adhesion Kinase
Authors: Sekine Yuichi , Tsuji Satoshi , Ikeda Osamu , Sugiyma Kenji , Oritani Kenji , Shimoda Kazuya , Muromoto Ryuta , Ohbayashi Norihiko , Yoshimura Akihiko , Matsuda Tadashi
Published: April 2014

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin homology- and Src homology 2-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies demonstrated that STAP-2 binds to STAT3 and STAT5, and regulates their signaling pathways. In the present study, we find that STAP-2-deficient splenocytes or T cells exhibit enhanced cell adhesion to fibronectin after PMA treatment, and that STAP-2-deficient T cells contain the increased protein contents of focal adhesion kinase (FAK). Furthermore, overexpression of STAP-2 induces a dramatic decrease in the protein contents of FAK and integrin-mediated T cell adhesion to fibronectin in Jurkat T cells via the degradation of FAK. Regarding the mechanism for this effect, we found that STAP-2 associates with FAK and enhances its degradation, proteasome inhibitors block FAK degradation, and STAP-2 recruits an endogenous E3 ubiquitin ligase, Cbl, to FAK. These results reveal a novel regulation mechanism for integrin-mediated signaling in T cells via STAP-2, which directly interacts with and degrades FAK.
Fusobacterium nucleatumInduces Fetal Death in Mice via Stimulation of TLR4-Mediated Placental Inflammatory Response
Authors: Liu Hongqi , Redline Raymond W. , Han Yiping W.
Published: April 2014

Intrauterine infection plays a pivotal role in preterm birth (PTB) and is characterized by inflammation. Currently, there is no effective therapy available to treat or prevent bacterial-induced PTB. Using Fusobacterium nucleatum, a Gram-negative anaerobe frequently associated with PTB, as a model organism, the mechanism of intrauterine infection was investigated. Previously, it was shown that F. nucleatum induced preterm and term stillbirth in mice. Fusobacterial-induced placental infection was characterized by localized bacterial colonization, inflammation, and necrosis. In this study, F. nucleatum was shown to activate both TLR2 and TLR4 in vitro. In vivo, the fetal death rate was significantly reduced in TLR4-deficient mice (C57BL/6 TLR4−/− and C3H/HeJ (TLR4d/d)), but not in TLR2-deficient mice (C57BL/6 TLR2−/−), following F. nucleatum infection. The reduced fetal death in TLR4-deficient mice was accompanied by decreased placental necroinflammatory responses in both C57BL/6 TLR4−/− and C3H/HeJ. Decreased bacterial colonization in the placenta was observed in C3H/HeJ, but not in C57BL/6 TLR4−/−. These results suggest that inflammation, rather than the bacteria per se, was the likely cause of fetal loss. TLR2 did not appear to be critically involved, as no difference in bacterial colonization, inflammation, or necrosis was observed between C57BL/6 and C57BL/6 TLR2−/− mice. A synthetic TLR4 antagonist, TLR4A, significantly reduced fusobacterial-induced fetal death and decidual necrosis without affecting the bacterial colonization in the placentas. TLR4A had no bactericidal activity nor did it affect the birth outcome in sham-infected mice. TLR4A could have promise as an anti-inflammatory agent for the treatment or prevention of bacterial-induced preterm birth.
Porphyromonas gingivalisSelectively Up-Regulates the HIV-1 Coreceptor CCR5 in Oral Keratinocytes
Authors: Giacaman Rodrigo A. , Nobbs Angela H. , Ross Karen F. , Herzberg Mark C.
Published: April 2014

Primary infection of oral epithelial cells by HIV-1, if it occurs, could promote systemic infection. Most primary systemic infections are associated with R5-type HIV-1 targeting the R5-specific coreceptor CCR5, which is not usually expressed on oral keratinocytes. Because coinfection with other microbes has been suggested to modulate cellular infection by HIV-1, we hypothesized that oral keratinocytes may up-regulate CCR5 in response to the oral endogenous pathogen Porphyromonas gingivalis by cysteine-protease (gingipains) activation of the protease-activated receptors (PARs) or LPS signaling through the TLRs. The OKF6/TERT-2-immortalized normal human oral keratinocyte line expressed CXCR4, whereas CCR5 was not detectable. When exposed to P. gingivalis ATCC 33277, TERT-2 cells induced greater time-dependent expression of CCR5-specific mRNA and surface coreceptors than CXCR4. By comparing arg- (Rgp) and lys-gingipain (Kgp) mutants, a mutant deficient in both proteases, and the action of trypsin, P. gingivalis Rgp was strongly suggested to cleave PAR-1 and PAR-2 to up-regulate CCR5. CCR5 was also slightly up-regulated by an isogenic gingipain-deficient mutant, suggesting the presence of a nongingipain-mediated mechanism. Purified P. gingivalis LPS also up-regulated CCR5. Blocking TLR2 and TLR4 receptors with Abs attenuated induction of CCR5, suggesting LPS signaling through TLRs. P. gingivalis, therefore, selectively up-regulated CCR5 by two independent signaling pathways, Rgp acting on PAR-1 and PAR-2, and LPS on TLR2 and TLR4. By inducing CCR5 expression, P. gingivalis coinfection could promote selective R5-type HIV-1 infection of oral keratinocytes.
Cellular and Humoral Autoreactivity in Idiopathic Pulmonary Fibrosis
Authors: Feghali-Bostwick Carol A. , Tsai Christopher G. , Valentine Vincent G. , Kantrow Stephen , Stoner Michael W. , Pilewski Joseph M. , Gadgil Aneal , George M. Patricia , Gibson Kevin F. , Choi Augustine M. K. , Kaminski Naftali , Zhang Yingze , Duncan Steven R.
Published: April 2014

Idiopathic pulmonary fibrosis (IPF) is a morbid, refractory lung disorder with an unknown pathogenesis. To investigate potential adaptive immune mechanisms in IPF, we compared phenotypes and effector functions of peripheral CD4 T cells, autoantibody production, and proliferative responses of pulmonary hilar lymph node CD4 T cells to autologous lung extracts from afflicted patients and normals. Our results show that greater proportions of peripheral CD4 T lymphocytes in IPF subjects expressed MHC class II and CD154 (CD40L), and they more frequently elaborated TGF-β1, IL-10, and TNF-α. Abnormal CD4 T cell clonal expansions were found in all IPF patients, and 82% of these subjects also had IgG autoantibodies against cellular Ags. IPF lung extracts stimulated proliferations of autologous CD4 T cells, unlike preparations from normals or those with other lung diseases, and the IPF proliferative responses were enhanced by repeated cycles of stimulation. Thus, CD4 T cells from IPF patients have characteristics typical of cell-mediated pathologic responses, including augmented effector functions, provision of facultative help for autoantibody production, oligoclonal expansions, and proliferations driven by an Ag present in diseased tissues. Recognition that an autoreactive immune process is present in IPF can productively focus efforts toward identifying the responsible Ag, and implementing more effective therapies.
Fully MHC-Disparate Mixed Hemopoietic Chimeras Show Specific Defects in the Control of Chronic Viral Infections
Authors: Koehn Brent H. , Williams Matthew A. , Borom Keshawna , Gangappa Shivaprakash , Pearson Thomas C. , Ahmed Rafi , Larsen Christian P.
Published: April 2014

The establishment of mixed allogeneic chimerism can induce donor-specific transplantation tolerance across full MHC barriers. However, a theoretical disadvantage of this approach is the possibility that the state of mixed chimerism might negatively affect the recipient’s immune competence to control pathogens. Previous studies using murine models have not supported this hypothesis, because they indicate that acute viral infections are cleared by chimeric animals with similar kinetics to that of unmanipulated controls. However, chronic or persistent viral infections often require a more complex and sustained response with cooperation between CD4 Th cells, CTL, and B cells for effective control. The current study indicates that profound defects become manifest in the control of chronic pathogenic infections in MHC-disparate mixed allogeneic chimeric mice. Furthermore, we show that ineffective priming of the donor-restricted CTL response leads to virus persistence, as well as severe T cell exhaustion. Our results further suggest that either T cell adoptive immunotherapy or selected MHC haplotype matching partially restore immune competence. These approaches may facilitate the translation of mixed chimerism therapeutic regimens.
Induction of Primary Biliary Cirrhosis in Guinea Pigs following Chemical Xenobiotic Immunization
Authors: Leung Patrick S. C. , Park Ogyi , Tsuneyama Koichi , Kurth Mark J. , Lam Kit S. , Ansari Aftab A. , Coppel Ross L. , Gershwin M. Eric
Published: April 2014

Although significant advances have been made in dissecting the effector mechanisms in autoimmunity, the major stumbling block remains defining the etiological events that precede disease. Primary biliary cirrhosis (PBC) illustrates this paradigm because of its high degree of heritability, its female predominance, and its extraordinarily specific and defined immune response and target destruction. In PBC, the major autoantigens belong to E2 components of the 2-oxo-acid dehydrogenase family of mitochondrially located enzymes that share a lipoylated peptide sequence that is the immunodominant target. Our previous work has demonstrated that synthetic mimics of the lipoate molecule such as 6-bromohexoanate demonstrate a high degree of reactivity with PBC sera prompted us to immunize groups of guinea pigs with 6-bromohexoanate conjugated to BSA. In this study, we provide serologic and immunohistochemical evidence that such immunized guinea pigs not only develop antimitochondrial autoantibody responses similar to human PBC, but also develop autoimmune cholangitis after 18 mo. Xenobiotic-immunized guinea pigs are the first induced model of PBC and suggest an etiology that has implications for the causation of other human autoimmune diseases. The data also reflect the likelihood that, in PBC, the multilineage antimitochondrial response is a pathogenic mechanism and that loss of tolerance and subsequent development of biliary lesions depends on either modification of the host mitochondrial Ag or a similar breakdown due to molecular mimicry.
Cutting Edge: Complement (C3d)-Linked Antigens Break B Cell Anergy
Authors: Lyubchenko Taras , Dal Porto Joseph M. , Holers V. Michael , Cambier John C.
Published: April 2014

C3dg adducts of Ag can coligate complement receptor type 2 (CR2; CD21) and the B cell Ag receptor. This interaction significantly amplifies BCR-mediated signals in Ag-naive wild-type mice, lowering the threshold for B cell activation and the generation of humoral immune responses as much as 1000-fold. In this study we demonstrate that CR2-mediated complementation of BCR signals can also overcome B cell anergy. Unlike Ag alone, BCR/CR2 costimulation (Ars-CCG/C3dg complexes) of anergic Ars/A1 B cells led to Ca2+ mobilization in vitro and the production of autoantibodies in vivo. Interestingly, the in vivo immune response of anergic cells occurs without the formation of germinal centers. These results suggest that the Ag unresponsiveness of anergic B cells can be overcome by cross-reactive (self-mimicking) Ags that have been complement-opsonized. This mechanism may place individuals exposed to complement-fixing bacteria at risk for autoimmunity.
CD154 Tone Sets the Signaling Pathways and Transcriptome Generated in Model CD40-Pluricompetent L3055 Burkitt’s Lymphoma Cells
Authors: Stewart Ross , Wei Wenbin , Challa Anita , Armitage Richard J. , Arrand John R. , Rowe Martin , Young Lawrence S. , Eliopoulos Aristides , Gordon John
Published: April 2014

Activated B cells reacting to small amounts of CD40L (CD154) maintain homeostasis by suppressing default apoptosis. Additional outcomes, particularly differentiation, demand higher CD40 occupancy. Here, focusing on survival, we compared changes in the transcriptome of pleiotropically competent, early passage L3055 Burkitt’s lymphoma cells confronted with low (picomolar) and high (nanomolar) concentrations of CD154 to gain insight into how a single receptor sets these distinct phenotypes. Of 267 genes altering transcriptional activity in response to strong CD154 tone, only 25 changed coordinately on low receptor occupancy. Seven of the top nine common up-regulated genes were targets of NF-κB. Direct measurement and functional inhibition of the NF-κB pathway revealed it to be central to a CD40-dependent survival signature. Although the canonical NF-κB axis was engaged by both signaling strengths equally, robust alternative pathway activation was a feature selective to a strong CD40 signal. Discriminatory exploitation of the two separate arms of NF-κB activation may indicate a principle whereby a cell senses and reacts differentially to shifting ligand availability. Identifying components selectively coupling CD40 to each axis could indicate targets for disruption in B cell pathologies underpinned by ectopic and/or hyper-CD154 activity such as neoplasia and some autoimmunities.
Proteinase-Activated Receptor-2 Promotes Allergic Sensitization to an Inhaled Antigen through a TNF-Mediated Pathway
Authors: Ebeling Cory , Lam Tong , Gordon John R. , Hollenberg Morley D. , Vliagoftis Harissios
Published: April 2014

The reason why particular inhaled Ags induce allergic sensitization while others lead to immune tolerance is unclear. Along with a genetic predisposition to atopy, intrinsic characteristics of these Ags must be important. A common characteristic of many allergens is that they either possess proteinase activity or are inhaled in particles rich in proteinases. Many allergens, such as house dust mite and cockroach allergens, have the potential to activate the proteinase-activated receptor (PAR)-2. In this study, we report that PAR-2 activation in the airways at the same time as exposure to inhaled Ags induces allergic sensitization, whereas exposure to Ag alone induces tolerance. BALB/c mice were administered OVA with a PAR-2 activating peptide intranasally. Upon allergen re-exposure mice developed airway inflammation and airway hyperresponsiveness, as well as OVA-specific T cells with a Th2 cytokine profile when restimulated with OVA in vitro. Conversely, mice given OVA alone or OVA with a PAR-2 control peptide developed tolerance. These tolerant mice did not develop airway inflammation or airway hyperresponsiveness, and developed OVA-specific T cells that secreted high levels of IL-10 when restimulated with OVA in vitro. Furthermore, pulmonary dendritic cell trafficking was altered in mice following intranasal PAR-2 activation. Finally, we showed that PAR-2-mediated allergic sensitization was TNF-dependent. Thus, PAR-2 activation in the airways could be a critical factor in the development of allergic sensitization following mucosal exposure to allergens with serine proteinase activity. Interfering with this pathway may prove to be useful for the prevention or treatment of allergic diseases.
Identification of an IL-7-Dependent Pre-T Committed Population in the Spleen
Authors: Gautreau Laetitia , Arcangeli Marie-Laure , Pasqualetto Valérie , Joret Anne-Marie , Garcia-Cordier Corinne , Mégret Jérôme , Schneider Elke , Ezine Sophie
Published: April 2014

Several extrathymic T cell progenitors have been described but their various contributions to the T cell lineage puzzle are unclear. In this study, we provide evidence for a splenic Lin−Thy1.2+ T cell-committed population, rare in B6 mice, abundant in TCRα−/−, CD3ε−/−, and nude mice, and absent in IL-7- and Rag-2-deficient mice. Neither B nor myeloid cells are generated in vivo and in vitro. The incidence of these pre-T cells is under the control of thymus and/or mature T cells, as revealed by graft experiments. Indeed, IL-7 consumption by mature T cells inhibits the growth of these pre-T cells. Moreover, the nude spleen contains an additional Lin−Thy1.2+CD25+ subset which is detected in B6 mice only after thymectomy. We establish that the full pre-T cell potential and proliferation capacity are only present in the c-kitlow fraction of progenitors. We also show that most CCR9+ progenitors are retained in the spleen of nude mice, but present in the blood of B6 mice. Thus, our data describe a new T cell lineage restricted subset that accumulates in the spleen before migration to the thymus.
Severe Depletion of Mucosal CD4+ T Cells in AIDS-Free Simian Immunodeficiency Virus-Infected Sooty Mangabeys
Authors: Gordon Shari N. , Klatt Nichole R. , Bosinger Steven E. , Brenchley Jason M. , Milush Jeffrey M. , Engram Jessica C. , Dunham Richard M. , Paiardini Mirko , Klucking Sara , Danesh Ali , Strobert Elizabeth A. , Apetrei Cristian , Pandrea Ivona V. , Kelvin David , Douek Daniel C. , Staprans Silvija I. , Sodora Donald L. , Silvestri Guido
Published: April 2014

HIV-infected humans and SIV-infected rhesus macaques experience a rapid and dramatic loss of mucosal CD4+ T cells that is considered to be a key determinant of AIDS pathogenesis. In this study, we show that nonpathogenic SIV infection of sooty mangabeys (SMs), a natural host species for SIV, is also associated with an early, severe, and persistent depletion of memory CD4+ T cells from the intestinal and respiratory mucosa. Importantly, the kinetics of the loss of mucosal CD4+ T cells in SMs is similar to that of SIVmac239-infected rhesus macaques. Although the nonpathogenic SIV infection of SMs induces the same pattern of mucosal target cell depletion observed during pathogenic HIV/SIV infections, the depletion in SMs occurs in the context of limited local and systemic immune activation and can be reverted if virus replication is suppressed by antiretroviral treatment. These results indicate that a profound depletion of mucosal CD4+ T cells is not sufficient per se to induce loss of mucosal immunity and disease progression during a primate lentiviral infection. We propose that, in the disease-resistant SIV-infected SMs, evolutionary adaptation to both preserve immune function with fewer mucosal CD4+ T cells and attenuate the immune activation that follows acute viral infection protect these animals from progressing to AIDS.
Polymorphisms in Genes Involved in Innate Immunity Predispose Toward Mycetoma Susceptibility
Authors: van de Sande Wendy W. J. , Fahal Ahmed , Verbrugh Henri , van Belkum Alex
Published: April 2014

Madurella mycetomatis is the main causative agent of mycetoma, a tumorous fungal infection characterized by the infiltration of large numbers of neutrophils at the site of infection. In endemic areas the majority of inhabitants have Abs to M. mycetomatis, although only a small proportion of individuals actually develop mycetomal disease. It therefore appears that neutrophils are unable to clear the infection in some individuals. To test this hypothesis, 11 single nucleotide polymorphisms involved in neutrophil function were studied in a population of Sudanese mycetoma patients vs geographically and ethnically matched controls. Significant differences in allele distribution for IL-8 (CXCL8), its receptor CXCR2, thrombospondin-4 (TSP-4), NO synthase 2 (NOS2), and complement receptor 1 (CR1) were found. Further, the NOS2Lambaréné polymorphism was clearly associated with lesion size. The genotypes obtained for CXCL8, its receptor CXCR2, and TSP-4 all predisposed to a higher CXCL8 expression in patients, which was supported by the detection of significantly elevated levels of CXCL8 in patient serum. The NOS2 genotype observed in healthy controls was correlated with an increase in NOS2 expression and higher concentrations of nitrate and nitrite in control serum. We present the first evidence of human genetic predisposition toward susceptibility to mycetoma, a neglected infection of the poor.
A Role for Dietary Selenium and Selenoproteins in Allergic Airway Inflammation
Authors: Hoffmann Peter R. , Jourdan-Le Saux Claude , Hoffmann Fukun W. , Chang Peter S. , Bollt Oana , He Qingping , Tam Elizabeth K. , Berry Marla J.
Published: April 2014

Asthma is driven by allergic airway inflammation and involves increased levels of oxidative stress. This has led to speculation that antioxidants like selenium (Se) may play important roles in preventing or treating asthma. We fed diets containing low (0.08 parts per million), medium (0.25 parts per million), or high (2.7 parts per million) Se to female C57BL/6 mice and used an established OVA challenge protocol to determine the relationship between Se intake and the development of allergic airway inflammation. Results demonstrated that mice fed medium levels of Se had robust responses to OVA challenge in the lung as measured by lung cytokine levels, airway cellular infiltrate, eosinophilia, serum anti-OVA IgE, airway hyperreactivity, goblet cell hyperplasia, and phosphorylated STAT-6 levels in the lung. In contrast, responses to OVA challenge were less robust in mice fed low or high levels of Se. In particular, mice fed low Se chow showed significantly lower responses compared with mice fed medium Se chow for nearly all readouts. We also found that within the medium Se group the expression of lung glutathione peroxidase-1 and liver selenoprotein P were increased in OVA-challenged mice compared with PBS controls. These data suggest that Se intake and allergic airway inflammation are not related in a simple dose-response manner, which may explain the inconsistent results obtained from previous descriptive studies in humans. Also, our results suggest that certain selenoproteins may be induced in response to Ag challenges within the lung.
Effects of Gamma Radiation on FcεRI and TLR-Mediated Mast Cell Activation
Authors: Soule Benjamin P. , Brown Jared M. , Kushnir-Sukhov Nataliya M. , Simone Nicole L. , Mitchell James B. , Metcalfe Dean D.
Published: April 2014

Ionizing gamma radiation has several therapeutic indications including bone marrow transplantation and tumor ablation. Among immune cells, susceptibility of lymphocytes to gamma radiation is well known. However, there is little information on the effects of gamma radiation on mast cells, which are important in both innate and acquired immunity. Previous studies have suggested that mast cells may release histamine in response to high doses of gamma radiation, whereas other reports suggest that mast cells are relatively radioresistant. No strong link has been established between gamma radiation and its effect on mast cell survival and activation. We examined both human and murine mast cell survival and activation, including mechanisms related to innate and acquired immune responses following gamma radiation. Data revealed that human and murine mast cells were resistant to gamma radiation-induced cytotoxicity and, importantly, that irradiation did not directly induce β-hexosaminidase release. Instead, a transient attenuation of IgE-mediated β-hexosaminidase release and cytokine production was observed which appeared to be the result of reactive oxygen species formation after irradiation. Mast cells retained the ability to phagocytose Escherichia coli particles and respond to TLR ligands as measured by cytokine production after irradiation. In vivo, there was no decrease in mast cell numbers in skin of irradiated mice. Additionally, mast cells retained the ability to respond to Ag in vivo as measured by passive cutaneous anaphylaxis in mice after irradiation. Mast cells are thus resistant to the cytotoxic effects and alterations in function after irradiation and, despite a transient inhibition, ultimately respond to innate and acquired immune activation signals.
Urokinase-Type Plasminogen Activator Stimulation of Monocyte Matrix Metalloproteinase-1 Production Is Mediated by Plasmin-Dependent Signaling through Annexin A2 and Inhibited by Inactive Plasmin
Authors: Zhang Yahong , Zhou Zhao-Hua , Bugge Thomas H. , Wahl Larry M.
Published: April 2014

Chronic inflammatory diseases are associated with connective tissue turnover that involves a series of proteases, which include the plasminogen activation system and the family of matrix metalloproteinases (MMPs). Urokinase-type plasminogen activator (uPA) and plasmin, in addition to their role in fibrinolysis and activation of pro-MMPs, have been shown to transduce intracellular signals through specific receptors. The potential for uPA and plasmin to also contribute to connective tissue turnover by directly regulating MMP production was examined in human monocytes. Both catalytically active high m.w. uPA, which binds to the uPAR, and low m.w. uPA, which does not, significantly enhanced MMP-1 synthesis by activated human monocytes. In contrast, the N-terminal fragment of uPA, which binds to uPAR, but lacks the catalytic site, failed to induce MMP-1 production, indicating that uPA-stimulated MMP-1 synthesis was plasmin dependent. Endogenous plasmin generated by the action of uPA or exogenous plasmin increased MMP-1 synthesis by signaling through annexin A2, as demonstrated by inhibition of MMP-1 production with Abs against annexin A2 and S100A10, a dimeric protein associated with annexin A2. Interaction of plasmin with annexin A2 resulted in the stimulation of ERK1/2 and p38 MAPK, cyclooxygenase-2, and PGE2, leading to increased MMP-1 production. Furthermore, binding of inactive plasmin to annexin A2 inhibited plasmin induction of MMP-1, suggesting that inactive plasmin may be useful in suppressing inflammation.
“Cytokine Storm” in the Phase I Trial of Monoclonal Antibody TGN1412: Better Understanding the Causes to Improve PreClinical Testing of Immunotherapeutics
Authors: Stebbings Richard , Findlay Lucy , Edwards Cherry , Eastwood David , Bird Chris , North David , Mistry Yogesh , Dilger Paula , Liefooghe Emily , Cludts Isabelle , Fox Bernard , Tarrant Gill , Robinson Jane , Meager Tony , Dolman Carl , Thorpe Susan J. , Bristow Adrian , Wadhwa Meenu , Thorpe Robin , Poole Stephen
Published: April 2014

The CD28-specific mAb TGN1412 rapidly caused a life-threatening “cytokine storm” in all six healthy volunteers in the Phase I clinical trial of this superagonist, signaling a failure of preclinical safety testing. We report novel in vitro procedures in which TGN1412, immobilized in various ways, is presented to human white blood cells in a manner that stimulates the striking release of cytokines and profound lymphocyte proliferation that occurred in vivo in humans. The novel procedures would have predicted the toxicity of this superagonist and are now being applied to emerging immunotherapeutics and to other therapeutics that have the potential to act upon the immune system. Data from these novel procedures, along with data from in vitro and in vivo studies in nonhuman primates, suggest that the dose of TGN1412 given to human volunteers was close to the maximum immunostimulatory dose and that TGN1412 is not a superagonist in nonhuman primates.
Enhancing Effect of IL-1α on Neurogenesis from Adult Human Mesenchymal Stem Cells: Implication for Inflammatory Mediators in Regenerative Medicine
Authors: Greco Steven J. , Rameshwar Pranela
Published: April 2014

Mesenchymal stem cells (MSCs) are mesoderm-derived cells, primarily resident in adult bone marrow. MSCs show lineage specificity in generating specialized cells such as stroma, fat, and cartilage. MSCs express MHC class II and function as phagocytes and APCs. Despite these immune-enhancing properties, MSCs also exert veto functions and show evidence for allogeneic transplantation. These properties, combined with ease in isolation and expansion, demonstrate MSCs as attractive candidates for tissue repair across allogeneic barriers. MSCs have also been shown to transdifferentiate in neuronal cells. We have reported expression of the neurotransmitter gene, Tac1, in MSC-derived neuronal cells, with no evidence of translation unless cells were stimulated with IL-1α. This result led us to question the potential role of immune mediators in the field of stem cell therapy. Using Tac1 as an experimental model, IL-1α was used as a prototypical inflammatory mediator to study functions on MSC-derived neuronal cells. Undifferentiated MSCs and those induced to form neurons were studied for their response to IL-1α and other proinflammatory cytokines using production of the major Tac1 peptide, substance P (SP), as readout. Although IL-1α induced high production of SP, a similar effect was not observed for all tested cytokines. The induced SP was capable of reuptake via its high-affinity NK1R and was found to stabilize IL-1R mRNA. IL-1α also enhanced the rate of neurogenesis, based on expression of neuronal markers and cRNA microarray analyses. The results provide evidence that inflammatory mediators need to be considered when deciding the course of MSC transplantation.
Natural Killer Cells in Perinatally HIV-1-Infected Children Exhibit Less Degranulation Compared to HIV-1-Exposed Uninfected Children and Their Expression of KIR2DL3, NKG2C, and NKp46 Correlates with Disease Severity
Authors: Ballan Wassim M. , Vu Bien-Aimee N. , Long Brian R. , Loo Christopher P. , Michaëlsson Jakob , Barbour Jason D. , Lanier Lewis L. , Wiznia Andrew A. , Abadi Jacobo , Fennelly Glenn J. , Rosenberg Michael G. , Nixon Douglas F.
Published: April 2014

NK cells play an integral role in the innate immune response by targeting virally infected and transformed cells with direct killing and providing help to adaptive responses through cytokine secretion. Whereas recent studies have focused on NK cells in HIV-1-infected adults, the role of NK cells in perinatally HIV-1-infected children is less studied. Using multiparametric flow cytometric analysis, we assessed the number, phenotype, and function of NK cell subsets in the peripheral blood of perinatally HIV-1-infected children on highly active antiretroviral therapy and compared them to perinatally exposed but uninfected children. We observed an increased frequency of NK cells expressing inhibitory killer Ig-like receptors in infected children. This difference existed despite comparable levels of total NK cells and NK cell subpopulations between the two groups. Additionally, NK cell subsets from infected children expressed, with and without stimulation, significantly lower levels of the degranulation marker CD107, which correlates with NK cell cytotoxicity. Lastly, increased expression of KIR2DL3, NKG2C, and NKp46 on NK cells correlated with decreased CD4+ T-lymphocyte percentage, an indicator of disease severity in HIV-1- infected children. Taken together, these results show that HIV-1-infected children retain a large population of cytotoxically dysfunctional NK cells relative to perinatally exposed uninfected children. This reduced function appears concurrently with distinct NK cell surface receptor expression and is associated with a loss of CD4+ T cells. This finding suggests that NK cells may have an important role in HIV-1 disease pathogenesis in HIV-1-infected children.
Cutting Edge: A Critical Role of Nitrogen Oxide in Preventing Inflammation upon Apoptotic Cell Clearance
Authors: Shibata Takehiko , Nagata Kisaburo , Kobayashi Yoshiro
Published: April 2014

Apoptotic cells are removed by phagocytes without causing inflammation. It remains largely unresolved whether anti-inflammatory mediators prevent neutrophil infiltration upon apoptotic cell clearance in vivo. In this study, we showed that, upon induction of apoptosis in the thymus by x-ray, inducible NO synthase knockout (KO) mice exhibited higher levels of neutrophil infiltration and production of MIP-2 and keratinocyte-derived chemokine (KC) in the thymus than wild-type (WT) mice. Furthermore, administration of NG-nitro-l-arginine methyl ester, an inhibitor of NO synthase, to x-irradiated WT mice increased the level of neutrophil infiltration to that of KO mice by the augmentation of MIP-2 and KC production. Additionally, thymic macrophages isolated from x-irradiated KO mice produced more MIP-2 and KC than those from WT mice. Thus, although apoptosis is believed to be noninflammatory, this is actually achieved by the production of immunosuppressive signals such as NO that counteract proinflammatory chemokines such as MIP-2 and KC.
Differential Effects of CpG DNA on IFN-β Induction and STAT1 Activation in Murine Macrophages versus Dendritic Cells: Alternatively Activated STAT1 Negatively Regulates TLR Signaling in Macrophages
Authors: Schroder Kate , Spille Martina , Pilz Andreas , Lattin Jane , Bode Konrad A. , Irvine Katharine M. , Burrows Allan D. , Ravasi Timothy , Weighardt Heike , Stacey Katryn J. , Decker Thomas , Hume David A. , Dalpke Alexander H. , Sweet Matthew J.
Published: April 2014

Classical STAT1 activation in response to TLR agonists occurs by phosphorylation of the Y701 and S727 residues through autocrine type I IFN signaling and p38 MAPK signaling, respectively. In this study, we report that the TLR9 agonist CpG DNA induced Ifn-β mRNA, as well as downstream type I IFN-dependent genes, in a MyD88-dependent manner in mouse myeloid dendritic cells. This pathway was required for maximal TNF and IL-6 secretion, as well as expression of cell surface costimulatory molecules. By contrast, neither A- nor B-type CpG-containing oligonucleotides induced Ifn-β in mouse bone marrow-derived macrophages (BMM) and a CpG-B oligonucleotide did not induce IFn-β in the macrophage-like cell line, J774. In BMM, STAT1 was alternatively activated (phosphorylated on S727, but not Y701), and was retained in the cytoplasm in response to CpG DNA. CpG DNA responses were altered in BMM from STAT1S727A mice; Il-12p40 and Cox-2 mRNAs were more highly induced, whereas Tlr4 and Tlr9 mRNAs were more repressed. The data suggest a novel inhibitory function for cytoplasmic STAT1 in response to TLR agonists that activate p38 MAPK but do not elicit type I IFN production. Indeed, the TLR7 agonist, R837, failed to induce Ifn-β mRNA and consequently triggered STAT1 phosphorylation on S727, but not Y701, in human monocyte-derived macrophages. The differential activation of Ifn-β and STAT1 by CpG DNA in mouse macrophages vs dendritic cells provides a likely mechanism for their divergent roles in priming the adaptive immune response.
Induction of the CTLA-4 Gene in Human Lymphocytes Is Dependent on NFAT Binding the Proximal Promoter
Authors: Gibson Heather M. , Hedgcock Carrie J. , Aufiero Barbara M. , Wilson Adam J. , Hafner Mikehl S. , Tsokos George C. , Wong Henry K.
Published: April 2014

CTLA-4 is a member of the costimulatory family, has homology to CD28, and binds the B7 family of ligands. Unlike CD28, CTLA-4 ligation transmits a negative signal in T cells. CTLA-4 expression, while inducible in most T cells, is expressed constitutively on T cells with a regulatory phenotype. The mechanism controlling CTLA-4 expression in human T cells is poorly characterized, thus we sought to better understand the mechanism of activation of the CTLA-4 gene. By cloning the 5′ upstream promoter and creating promoter-deletion reporter constructs, we show that the proximal promoter is critical for activating the CTLA-4 gene. Within this region, we identify a NFAT consensus sequence that binds NFAT with high affinity that differs from other NFAT sequences and does not recruit AP-1. Analysis of the chromatin proteins in the native CTLA-4 gene shows that this promoter region becomes associated with acetylated histones by chromatin immunoprecipitation assays. In addition, NFAT1 binds to the promoter of the CTLA-4 gene after stimulation by chromatin immunoprecipitation. The functional requirement of the NFAT site for CTLA-4 transcription was demonstrated by mutations in the NFAT site that abolished the activity of the promoter. Furthermore, inhibitors of NFAT suppressed CTLA-4 gene expression, indicating that NFAT plays a critical role in regulating the induction of the CTLA-4 gene in lymphocytes. The identification of NFAT as a critical regulator of the CTLA-4 gene suggests that targeting NFAT function may lead to novel approaches to modulate the CTLA-4 gene to control the immune response.
Involvement of Phosphatidylinositol 3-Kinase-Mediated Up-Regulation of IκBα in Anti-Inflammatory Effect of Gemfibrozil in Microglia
Authors: Jana Malabendu , Jana Arundhati , Liu Xiaojuan , Ghosh Sankar , Pahan Kalipada
Published: April 2014

The present study underlines the importance of PI3K in mediating the anti-inflammatory effect of gemfibrozil, a prescribed lipid-lowering drug for humans, in mouse microglia. Gemfibrozil inhibited LPS-induced expression of inducible NO synthase (iNOS) and proinflammatory cytokines in mouse BV-2 microglial cells and primary microglia. By overexpressing wild-type and dominant-negative constructs of peroxisome proliferator-activated receptor-α (PPAR-α) in microglial cells and isolating primary microglia from PPAR-α−/− mice, we have demonstrated that gemfibrozil inhibits the activation of microglia independent of PPAR-α. Interestingly, gemfibrozil induced the activation of p85α-associated PI3K (p110β but not p110α) and inhibition of that PI3K by either chemical inhibitors or dominant-negative mutants abrogated the inhibitory effect of gemfibrozil. Conversely, overexpression of the constitutively active mutant of p110 enhanced the inhibitory effect of gemfibrozil on LPS-induced expression of proinflammatory molecules. Similarly, gemfibrozil also inhibited fibrillar amyloid β (Aβ)-, prion peptide (PrP)-, dsRNA (poly IC)-, HIV-1 Tat-, and 1-methyl-4-phenylpyridinium (MPP+)-, but not IFN-γ-, induced microglial expression of iNOS. Inhibition of PI3K also abolished the inhibitory effect of gemfibrozil on Aβ-, PrP-, poly IC-, Tat-, and MPP+-induced microglial expression of iNOS. Involvement of NF-κB activation in LPS-, Aβ-, PrP-, poly IC-, Tat-, and MPP+-, but not IFN-γ-, induced microglial expression of iNOS and stimulation of IκBα expression and inhibition of NF-κB activation by gemfibrozil via the PI3K pathway suggests that gemfibrozil inhibits the activation of NF-κB and the expression of proinflammatory molecules in microglia via PI3K-mediated up-regulation of IκBα.
TGF-β1 Variants in Chronic Beryllium Disease and Sarcoidosis
Authors: Jonth Alexas C. , Silveira Lori , Fingerlin Tasha E. , Sato Hiroe , Luby Julie C. , Welsh Kenneth I. , Rose Cecile S. , Newman Lee S. , du Bois Roland M. , Maier Lisa A. ,   The ACCESS Group
Published: April 2014

Evidence suggests a genetic predisposition to chronic beryllium disease (CBD) and sarcoidosis, which are clinically and pathologically similar granulomatous lung diseases. TGF-β1, a cytokine involved in mediating the fibrotic/Th1 response, has several genetic variants which might predispose individuals to these lung diseases. We examined whether certain TGF-β1 variants and haplotypes are found at higher rates in CBD and sarcoidosis cases compared with controls and are associated with disease severity indicators for both diseases. Using DNA from sarcoidosis cases/controls from A Case Control Etiologic Study of Sarcoidosis Group (ACCESS) and CBD cases/controls, TGF-β1 variants were analyzed by sequence-specific primer PCR. No significant differences were found between cases and controls for either disease in the TGF-β1 variants or haplotypes. The −509C and codon 10T were significantly associated with disease severity indicators in both CBD and sarcoidosis. Haplotypes that included the −509C and codon 10T were also associated with more severe disease, whereas one or more copies of the haplotype containing the −509T and codon 10C was protective against severe disease for both sarcoidosis and CBD. These studies suggest that the −509C and codon 10T, implicated in lower levels of TGF-β1 protein production, are shared susceptibility factors associated with more severe granulomatous disease in sarcoidosis and CBD. This association may be due to lack of down-regulation by TGF-β1, although future studies will be needed to correlate TGF-β1 protein levels with known TGF-β1 genotypes and assess whether there is a shared mechanisms for TGF-β1 in these two granulomatous diseases.
Natural Phosphorylation of CD5 in Chronic Lymphocytic Leukemia B Cells and Analysis of CD5-Regulated Genes in a B Cell Line Suggest a Role for CD5 in Malignant Phenotype
Authors: Gary-Gouy Hélène , Sainz-Perez Alexander , Marteau Jean-Brice , Marfaing-Koka Anne , Delic Jozo , Merle-Beral Hélène , Galanaud Pierre , Dalloul Ali
Published: April 2014

Chronic lymphocytic leukemia (CLL) results in the accumulation of B cells, presumably reflecting the selection of malignant cell precursors with Ag combined with complex alterations in protein activity. Repeated BCR stimulation of normal B cells leads to anergy and CD5 expression, both of which are features of CLL. Because CD5 is phosphorylated on tyrosine following BCR engagement and negatively regulates BCR signaling in normal B cells, we investigated its phosphorylation status and found it to be naturally phosphorylated on tyrosine but not on serine residues in CLL samples. To analyze the role of CD5, we established a B cell line in which CD5 is phosphorylated. Gene profiling of vector vs CD5-transfected B cells pointed out gene groups whose expression was enhanced: Apoptosis inhibitors (BCL2), NF-κB (RELB, BCL3), Wnt, TGFβ, VEGF, MAPKs, Stats, cytokines, chemokines (IL-10, IL-10R, IL-2R, CCL-3, CCL-4, and CCR7), TLR-9, and the surface Ags CD52, CD54, CD70, and CD72. Most of these gene groups are strongly expressed in CLL B cells as compared with normal B cells. Unexpectedly, metabolic pathways, namely cholesterol synthesis and adipogenesis, are also enhanced by CD5. Conversely, CD5 inhibited genes involved in RNA splicing and processing, ribosome biogenesis, proteasome, and CD80 and CD86 Ags, whose expression is low in CLL. Comparison of CD5- vs tailless CD5-transfected cells further demonstrated the role of CD5 phosphorylation in the regulation of selected genes. These results support a model where CLL cells are chronically stimulated, leading to CD5 activation and cell survival. In addition to CD5 itself, we point to several CD5-induced genes as potential therapeutic targets.
Ubiquitinylation of Igβ Dictates the Endocytic Fate of the B Cell Antigen Receptor
Authors: Zhang Miao , Veselits Margaret , O’Neill Shannon , Hou Ping , Reddi Alagarsamy L. , Berlin Ilana , Ikeda Masato , Nash Piers D. , Longnecker Richard , Band Hamid , Clark Marcus R.
Published: April 2014

In both infection and autoimmunity, the development of high-affinity Abs and memory requires B cells to efficiently capture and process Ags for presentation to cognate T cells. Although a great deal is known about how Ags are processed, the molecular mechanisms by which the BCR captures Ag for processing are still obscure. In this study, we demonstrate that the Igβ component of the BCR is diubiquitinylated and that this is dependent on the E3 ligase Itch. Itch−/− B lymphocytes manifest both a defect in ligand-induced BCR internalization and endocytic trafficking to late endosomal Ag-processing compartments. In contrast, analysis of ubiquitinylation-defective receptors demonstrated that the attachment of ubiquitins to Igβ is required for endosomal sorting and for the presentation of Ag to T cells, yet, ubiquitinylation is dispensable for receptor internalization. Membrane-bound Igμ was not detectably ubiquitinylated nor were the conserved lysines in the mu cytosolic tail required for trafficking to late endosomes. These results demonstrate that ubiquitinylation of a singular substrate, Igβ, is required for a specific receptor trafficking event. However, they also reveal that E3 ligases play a broader role in multiple processes that determine the fate of Ag-engaged BCR complexes.
Inhibition of Terminal Complement Components in Presensitized Transplant Recipients Prevents Antibody-Mediated Rejection Leading to Long-Term Graft Survival and Accommodation
Authors: Wang Hao , Arp Jacqueline , Liu Weihua , Faas Susan J. , Jiang Jifu , Gies David R. , Ramcharran Siobhan , Garcia Bertha , Zhong Robert , Rother Russell P.
Published: April 2014

Ab-mediated rejection (AMR) remains the primary obstacle in presensitized patients following organ transplantation, as it is refractory to anti-T cell therapy and can lead to early graft loss. Complement plays an important role in the process of AMR. In the present study, a murine model was designed to mimic AMR in presensitized patients. This model was used to evaluate the effect of blocking the fifth complement component (C5) with an anti-C5 mAb on prevention of graft rejection. BALB/c recipients were presensitized with C3H donor skin grafts 7 days before heart transplantation from the same donor strain. Heart grafts, transplanted when circulating anti-donor IgG Abs were at peak levels, were rejected in 3 days. Graft rejection was characterized by microvascular thrombosis and extensive deposition of Ab and complement in the grafts, consistent with AMR. Anti-C5 administration completely blocked terminal complement activity and local C5 deposition, and in combination with cyclosporine and short-term cyclophosphamide treatment, it effectively prevented heart graft rejection. These recipients achieved permanent graft survival for >100 days with normal histology despite the presence of systemic and intragraft anti-donor Abs and complement, suggesting ongoing accommodation. Furthermore, double-transplant experiments demonstrated that immunological alterations in both the graft and the recipient were required for successful graft accommodation to occur. These data suggest that terminal complement blockade with a functionally blocking Ab represents a promising therapeutic approach to prevent AMR in presensitized recipients.