bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-7/Anti-IL-7 mAb Complexes Restore T Cell Development and Induce Homeostatic T Cell Expansion without Lymphopenia
Authors: Boyman Onur , Ramsey Chris , Kim David M. , Sprent Jonathan , Surh Charles D.
Published: April 2014

IL-7, a member of the common γ-chain family of cytokines, is essential for B and T lymphocyte development and homeostasis of mature T cell subsets. Thus, naive and memory T cells are both dependent on IL-7 for survival and homeostatic proliferation under lymphopenic conditions. In line with prior findings with IL-2, we show in this study that the biological activity of IL-7 in vivo is greatly increased by association with anti-IL-7 mAb. Under in vivo conditions, IL-7/mAb complexes displayed 50- to 100-fold higher activity than free IL-7 and induced massive expansion of pre-B cells. IL-7/mAb complexes also increased thymopoiesis in normal mice and restored thymopoeisis in IL-7-deficient mice. For mature T cells, IL-7/mAb complexes induced marked homeostatic proliferation of both naive and memory CD4+ and CD8+ cell subsets even under normal T cell-replete conditions. Finally, IL-7/mAb complexes were able to enhance the magnitude of the primary response of Ag-specific naive CD8+ cells. The strong stimulatory activity of IL-7/mAb complexes could be useful for treatment of immunodeficiency and cancer.
Circulating Myeloid Dendritic Cells of Advanced Cancer Patients Result in Reduced Activation and a Biased Cytokine Profile in Invariant NKT Cells
Authors: van der Vliet Hans J. J. , Wang Ruojie , Yue Simon C. , Koon Henry B. , Balk Steven P. , Exley Mark A.
Published: April 2014

CD1d-restricted invariant NKT (iNKT) cells play important regulatory roles in various immune responses, including antitumor immune responses. Previous studies have demonstrated quantitative and qualitative defects in iNKT cells of cancer patients, and these defects are clinically relevant as they are associated with poor prognosis. In this study we demonstrate that defects in the iNKT cell population can, at least in part, be attributed to defective interactions between iNKT cells and CD1d-expressing circulating myeloid dendritic cells (mDC), as mDC of patients with advanced melanoma and renal cell cancer reduced the activation and Th1 cytokine production of healthy donor-derived iNKT cells. Interestingly, this reduced activation of iNKT cells was restricted to patients with low circulating iNKT cell numbers and could be reversed by IL-12 and in part by the neutralization of TGF-β, but it was further reduced by the neutralization of IL-10 in vitro. Additional experiments revealed discordant roles for TGF-β and IL-10 on human iNKT cells, because TGF-β suppressed iNKT cell activation and proliferation and IFN-γ production while IL-10 was identified as a cytokine involved in stimulating the activation and expansion of iNKT cells that could subsequently suppress NK cell and T cell responses.
Regulatory CD4+ T Cells Are Crucial for Preventing CD8+ T Cell-Mediated Autoimmunity
Authors: Poitrasson-Rivière Maud , Bienvenu Boris , Le Campion Armelle , Bécourt Chantal , Martin Bruno , Lucas Bruno
Published: April 2014

In vivo studies have shown that regulatory CD4+ T cells regulate conventional CD4+ T cell responses to self- and environmental Ags. However, it remains unclear whether regulatory CD4+ T cells control CD8+ T cell responses to self, directly, or indirectly by decreasing available CD4+ T cell help. We have developed an experimental mouse model in which suppressive and helper T cells cannot mediate their functions. The mouse chimeras generated were not viable and rapidly developed multiple organ autoimmunity. These features were correlated with strong CD8+ T cell activation and accumulation in both lymphoid and nonlymphoid organs. In vivo Ab treatment and secondary transfer experiments demonstrated that regulatory CD4+ T cells play an important direct role in the prevention of peripheral CD8+ T cell-mediated autoimmunity.
Regulatory CD4+CD25+Foxp3+ T Cells Selectively Inhibit the Spontaneous Form of Lymphopenia-Induced Proliferation of Naive T Cells
Authors: Winstead Colleen J. , Fraser Joanne M. , Khoruts Alexander
Published: April 2014

Regulatory CD4+CD25+Foxp3+ T cells play a critical role in controlling autoimmunity and T cell homeostasis. However, their role in regulation of lymphopenia-induced proliferation (LIP), a potential mechanism for generation of autoaggressive T cells, has been poorly defined. Currently, two forms of LIP are recognized: spontaneous and homeostatic. Spontaneous LIP is characterized by fast, burst-like cell-cycle activity, and may allow effector T cell differentiation. Homeostatic LIP is characterized by slow and steady cell cycle activity and is not associated with the acquisition of an effector phenotype. In this study, we demonstrate that CD4+CD25+Foxp3+ T cells suppress the spontaneous, but not homeostatic, LIP of naive CD8 and CD4 T cells. However, selective inhibition of spontaneous LIP does not fully explain the tolerogenic role of Tregs in lymphopenia-associated autoimmunity. We show here that suppression of LIP in the lymphoid tissues is independent of Treg-derived IL-10. However, IL-10-deficient Tregs are partially defective in their ability to prevent colitis caused by adoptive transfer of CD4 T cells into RAG−/− mice. We propose that Tregs may inhibit emergence of effector T cells during the inductive phase of the immune response in the secondary lymphoid tissues by IL-10-independent mechanisms. In contrast, Treg-mediated inhibition of established effector T cells does require IL-10. Both Treg functions appear to be important in control of lymphopenia-associated autoimmunity.
Regulatory Role of B Cells in a Murine Model of Allergic Airway Disease
Authors: Singh Anurag , Carson William F. , Secor Eric R. , Guernsey Linda A. , Flavell Richard A. , Clark Robert B. , Thrall Roger S. , Schramm Craig M.
Published: April 2014

Mice sensitized to OVA and subjected to acute OVA aerosol exposures develop allergic airway disease (AAD). However, chronic continuous Ag exposure results in resolution of AAD and the development of local inhalational tolerance (LIT). Because we have previously observed the persistence of B cells in the bronchoalveolar lavage (BAL) and hilar lymph nodes (HLN) at the resolution stage of this model, we investigated the role of B cells in the modulation of AAD. Although B cell-deficient mice developed LIT, adoptive transfer of HLN B cells from LIT mice to OVA-sensitized recipients resulted in attenuated AAD following subsequent OVA aerosol exposure, as determined by reduced BAL leukocytosis and eosinophilia, decreased tissue inflammation, and absent methacholine hyper-responsiveness. In similar adoptive transfer studies, HLN B cells from AAD mice were without effect. The protection transferred by LIT HLN B cells was Ag specific and was associated with accumulation of Foxp3+ T regulatory cells regionally in BAL and HLN, but not systemically in the spleen. Fluorescent labeling of LIT HLN B cells before adoptive transfer demonstrated that these cells had the capacity to migrate to local inflammatory sites. In vitro assessment demonstrated that the LIT HLN B cells exerted this regulatory effect via TGF-β induced conversion of CD4+CD25− T effector cells into functionally suppressive CD4+CD25+Foxp3+ T regulatory cells. These findings illustrated a novel regulatory role for regional B cells in AAD and suggested a possible contributory role of B cells, along with other cell types, in the establishment of LIT.
Molecular and Immunological Characterization of a Wheat Serine Proteinase Inhibitor as a Novel Allergen in Baker's Asthma
Authors: Constantin Claudia , Quirce Santiago , Grote Monika , Touraev Alisher , Swoboda Ines , Stoecklinger Angelika , Mari Adriano , Thalhamer Josef , Heberle-Bors Erwin , Valenta Rudolf
Published: April 2014

IgE-mediated sensitization to wheat flour belongs to the most frequent causes of occupational asthma. A cDNA library from wheat seeds was constructed and screened with serum IgE from baker's asthma patients. One IgE-reactive phage clone contained a full-length cDNA coding for an allergen with a molecular mass of 9.9 kDa and an isoelectric point of 6. According to sequence analysis it represents a member of the potato inhibitor I family, a group of serine proteinase inhibitors, and thus is the first allergen belonging to the group 6 pathogenesis-related proteins. The recombinant wheat seed proteinase inhibitor was expressed in Escherichia coli and purified to homogeneity. According to circular dichroism analysis, it represented a soluble and folded protein with high thermal stability containing mainly β-sheets, random coils, and an α-helical element. The recombinant allergen showed allergenic activity in basophil histamine release assays and reacted specifically with IgE from 3 of 22 baker's asthma patients, but not with IgE from grass pollen allergic patients or patients suffering from food allergy to wheat. Allergen-specific Abs were raised to localize the allergen by immunogold electron microscopy in the starchy endosperm and the aleuron layer. The allergen is mainly expressed in mature wheat seeds and, despite an ∼50% sequence identity, showed no relevant cross-reactivity with allergens from other plant-derived food sources such as maize, rice, beans, or potatoes. Recombinant wheat serine proteinase inhibitor, when used in combination with other specific allergens, may be useful for the diagnosis and therapy of IgE-mediated baker's asthma.
Anthrax Lethal Toxin Enhances TNF-Induced Endothelial VCAM-1 Expression via an IFN Regulatory Factor-1-Dependent Mechanism
Authors: Warfel Jason M. , D'Agnillo Felice
Published: April 2014

Impaired host defenses and vascular dysfunction are hallmarks of the late, antibiotic-refractory stages of systemic anthrax infection. Anthrax lethal toxin (LT), a key virulence factor of Bacillus anthracis, was previously shown to enhance VCAM-1 expression on primary human endothelial cells suggesting a causative link between dysregulated adhesion molecule expression and the poor immune response and vasculitis associated with anthrax. In this study, we report that LT amplification of TNF-induced VCAM-1 expression is driven transcriptionally by the cooperative activation of NF-κB and IFN regulatory factor-1 (IRF-1). LT enhancement of NF-κB phosphorylation and nuclear translocation correlated temporally with a delayed reaccumulation of IκBα, while increased induction of IRF-1 was linked to STAT1 activation. LT failed to augment TNF-induced ICAM-1 or E-selectin expression, two adhesion molecules regulated by NF-κB, but not IRF-1. These results suggest that LT can differentially modulate NF-κB target genes and highlight the importance of IRF-1 in VCAM-1 enhancement. Altering the activity of key transcription factors involved in host response to infection may be a critical mechanism by which LT contributes to anthrax pathogenesis.
Murine Bactericidal/Permeability-Increasing Protein Inhibits the Endotoxic Activity of Lipopolysaccharide and Gram-Negative Bacteria
Authors: Wittmann Irene , Schönefeld Marc , Aichele Diana , Groer Gerhard , Gessner André , Schnare Markus
Published: April 2014

Recognition of LPS by TLR4 initiates inflammatory responses inducing potent antimicrobial immunity. However, uncontrolled inflammatory responses can be detrimental. To prevent the development of septic shock during an infection with Gram-negative bacteria, the immune system has developed mechanisms to neutralize LPS by specialized proteins. In this study, we report the recombinant expression and functional characterization of the mouse homolog of human bactericidal/permeability-increasing protein (BPI). Purified recombinant mouse BPI was able to neutralize LPS-mediated activation of macrophages and to block LPS-dependent maturation of dendritic cells. Recombinant mouse BPI neutralized the capacity of Gram-negative bacteria to activate immune cells, but did not influence the stimulatory properties of Gram-positive bacteria. Unlike human BPI, mouse BPI failed to kill or inhibit the growth of Pseudomonas aeruginosa. Together, these data demonstrate that murine BPI is a potent LPS-neutralizing protein that may limit innate immune responses during Gram-negative infections.
Administration of a Synthetic TLR4 Agonist Protects Mice from Pneumonic Tularemia
Authors: Lembo Annalisa , Pelletier Mark , Iyer Ravi , Timko Michele , Dudda Jan C. , West T. Eoin , Wilson Christopher B. , Hajjar Adeline M. , Skerrett Shawn J.
Published: April 2014

Francisella tularensis is a Gram-negative intracellular pathogen that causes the zoonosis tularemia. Because F. tularensis LPS causes weak TLR4 activation, we hypothesized that administration of a synthetic TLR4 agonist, aminoalkyl glucosaminide phosphate (AGP), would boost the innate immune system and compensate for reduced TLR4 stimulation. Intranasal administration of AGPs induced intrapulmonary production of proinflammatory cytokines and chemokines. Mice treated with AGPs before and after inhalation of Francisella novicida exhibited augmented cytokine and inflammatory responses to infection; reduced bacterial replication in lung, liver, and spleen; and increased survival, whereas all PBS-treated control mice died within 4 days of infection, all AGP-treated mice showed prolonged time-to-death, and 30–60% of AGP-treated mice survived. The protective effect of AGP was lost in mice lacking IFN-γ. Long-term survivors developed specific Th1 splenocyte responses and specific Abs dominated by IgG2 isotypes. Survivors were fully protected from rechallenge with aerosolized F. novicida. Thus, preventive administration of AGP successfully modulated innate immune responses to aerosolized F. novicida, leading to protective immunity to pneumonic tularemia. This is the first report of the protective effect of a TLR ligand on resistance to F. novicida-induced pneumonic tularemia.
Tissue Transglutaminase Activation Modulates Inflammation in Cystic Fibrosis via PPARγ Down-Regulation
Authors: Maiuri Luigi , Luciani Alessandro , Giardino Ida , Raia Valeria , Villella Valeria R. , D'Apolito Maria , Pettoello-Mantovani Massimo , Guido Stefano , Ciacci Carolina , Cimmino Mariano , Cexus Olivier N. , Londei Marco , Quaratino Sonia
Published: April 2014

Cystic fibrosis (CF), the most common life-threatening inherited disease in Caucasians, is due to mutations in the CF transmembrane conductance regulator (CFTR) gene and is characterized by airways chronic inflammation and pulmonary infections. The inflammatory response is not secondary to the pulmonary infections. Indeed, several studies have shown an increased proinflammatory activity in the CF tissues, regardless of bacterial infections, because inflammation is similarly observed in CFTR-defective cell lines kept in sterile conditions. Despite recent studies that have indicated that CF airway epithelial cells can spontaneously initiate the inflammatory cascade, we still do not have a clear insight of the molecular mechanisms involved in this increased inflammatory response. In this study, to understand these mechanisms, we investigated ex vivo cultures of nasal polyp mucosal explants of CF patients and controls, CFTR-defective IB3-1 bronchial epithelial cells, C38 isogenic CFTR corrected, and 16HBE normal bronchial epithelial cell lines. We have shown that a defective CFTR induces a remarkable up-regulation of tissue transglutaminase (TG2) in both tissues and cell lines. The increased TG2 activity leads to functional sequestration of the anti-inflammatory peroxisome proliferator-activated receptor γ and increase of the classic parameters of inflammation, such as TNF-α, tyrosine phosphorylation, and MAPKs. Specific inhibition of TG2 was able to reinstate normal levels of peroxisome proliferator-activated receptor-γ and dampen down inflammation both in CF tissues and CFTR-defective cells. Our results highlight an unpredicted central role of TG2 in the mechanistic pathway of CF inflammation, also opening a possible new wave of therapies for sufferers of chronic inflammatory diseases.
Factor VIII Hydrolysis Mediated by Anti-Factor VIII Autoantibodies in Acquired Hemophilia
Authors: Wootla Bharath , Dasgupta Suryasarathi , Dimitrov Jordan D. , Bayry Jagadeesh , Lévesque Hervé , Borg Jeanne-Yvonne , Borel-Derlon Annie , Rao Desirazu N. , Friboulet Alain , Kaveri Srinivas V. , Lacroix-Desmazes Sébastien
Published: April 2014

Acquired hemophilia is a rare hemorrhagic disorder caused by the spontaneous appearance of inhibitory autoantibodies directed against endogenous coagulation factor VIII (FVIII). Inhibitory Abs also arise in patients with congenital hemophilia A as alloantibodies directed to therapeutic FVIII. Both autoimmune and alloimmune inhibitors neutralize FVIII by steric hindrance. We have described FVIII-hydrolyzing IgG in 50% of inhibitor-positive patients with severe hemophilia A that inactivate therapeutic FVIII. In this study, we investigated the presence of autoimmune FVIII-hydrolyzing IgG in patients with acquired hemophilia. Pooled IgG from healthy donors demonstrated moderate FVIII-hydrolyzing activity (56 ± 26 μmol/min/mol). Purified IgG from 21 of 45 patients with acquired hemophilia demonstrated FVIII hydrolysis rates (mean 219 ± 94 μmol/min/mol) significantly greater than that of control IgG. Three of four patients followed over the course of the disease had rates of FVIII hydrolysis that co-evolved with inhibitory titers in plasma, suggesting that IgG-mediated FVIII hydrolysis participates, in part, in FVIII inactivation. The present work extends the scope of the diseases associated with FVIII proteolysis and points toward the importance of FVIII as a key target substrate for hydrolytic immunoglobulins. Our data suggest that elevated levels of FVIII-hydrolyzing IgG in acquired hemophilia result from the exacerbation of a physiological catalytic immune response.
Human TCR That Incorporate CD3ζ Induce Highly Preferred Pairing between TCRα and β Chains following Gene Transfer
Authors: Sebestyén Zsolt , Schooten Erik , Sals Tamara , Zaldivar Irene , San José Esther , Alarcón Balbino , Bobisse Sara , Rosato Antonio , Szöllősi János , Gratama Jan Willem , Willemsen Ralph A. , Debets Reno
Published: April 2014

TCR gene therapy is adversely affected by newly formed TCRαβ heterodimers comprising exogenous and endogenous TCR chains that dilute expression of transgenic TCRαβ dimers and are potentially self-reactive. We have addressed TCR mispairing by using a modified two-chain TCR that encompasses total human CD3ζ with specificities for three different Ags. Transfer of either TCRα:CD3ζ or β:CD3ζ genes alone does not result in surface expression, whereas transfer of both modified TCR chains results in high surface expression, binding of peptide-MHC complexes and Ag-specific T cell functions. Genetic introduction of TCRαβ:ζ does not compromise surface expression and functions of an endogenous TCRαβ. Flow cytometry fluorescence resonance energy transfer and biochemical analyses demonstrate that TCRαβ:CD3ζ is the first strategy that results in highly preferred pairing between CD3ζ-modified TCRα and β chains as well as absence of TCR mispairing between TCR:CD3ζ and nonmodified TCR chains. Intracellular assembly and surface expression of TCR:CD3ζ chains is independent of endogenous CD3γ, δ, and ε. Taken together, our data support the use of TCRαβ:CD3ζ to prevent TCR mispairing, which may provide an adequate strategy to enhance efficacy and safety of TCR gene transfer.
HLA-DQ6 (DQB1*0601)-Restricted T Cells Protect against Experimental Autoimmune Encephalomyelitis in HLA-DR3.DQ6 Double-Transgenic Mice by Generating Anti-Inflammatory IFN-γ
Authors: Mangalam Ashutosh , Luckey David , Basal Eati , Behrens Marshall , Rodriguez Moses , David Chella
Published: April 2014

The human MHC class II genes are associated with genetic susceptibility to multiple sclerosis (MS), a chronic inflammatory demyelinating disease of the CNS of presumed autoimmune origin. These genes encode for proteins responsible for shaping immune response. The exact role of HLA-DQ and -DR genes in disease pathogenesis is not well-understood due to the high polymorphism, linkage disequilibrium, and heterogeneity of human populations. The advent of HLA class II-transgenic (Tg) mice has helped in answering some of these questions. Previously, using single-Tg mice (expressing the HLA-DR or -DQ gene), we showed that proteolipid protein (PLP)91–110 peptide induced classical experimental autoimmune encephalomyelitis only in DR3.Aβ° mice, suggesting that DR3 (DRB1*0301) is a disease susceptible gene in the context of PLP. Human population studies have suggested that HLA-DQ6 (DQB1*0601) may be a protective gene in MS. To test this disease protection in an experimental model, we generated double-Tg mice expressing both HLA-DR3 and -DQ6. Introduction of DQ6 onto DR3-Tg mice led to a decrease in disease incidence on immunization with PLP91–110 peptide indicating a dominant protective role of DQ6. This protective effect is due to high levels of IFN-γ produced by DQ6-restricted T cells, which suppressed proliferation of encephalitogenic DR3-restricted T cells by inducing apoptosis. Our study indicates that DQ6 modifies the PLP91–110-specific T cell response in DR3 through anti-inflammatory effects of IFN-γ, which is protective for experimental autoimmune encephalomyelitis. Thus, our double-Tg mouse provides a novel model in which to study epistatic interactions between HLA class II molecules in MS.
Innate Immunity Mediated by TLR5 as a Novel Antiinflammatory Target for Cystic Fibrosis Lung Disease
Authors: Blohmke Christoph J. , Victor Rachel E. , Hirschfeld Aaron F. , Elias Isaac M. , Hancock David G. , Lane Cheryl R. , Davidson A. George F. , Wilcox Pearce G. , Smith Kelly D. , Overhage Joerg , Hancock Robert E. W. , Turvey Stuart E.
Published: April 2014

Novel therapies to target lung inflammation are predicted to improve the lives of people with cystic fibrosis (CF) but specific antiinflammatory targets have not been identified. The goal of this study was to establish whether TLR5 signaling is the key molecular pathway mediating lung inflammation in CF, and to determine whether strategies to inhibit TLR5 can reduce the damaging inflammatory response. The innate immune responses were analyzed in both airway epithelial cells and primary PBMCs from CF patients and matched controls. Additionally, 151 clinical isolates of Pseudomonas aeruginosa from CF patients were assessed for motility and capacity to activate TLR5. Blood and airway cells from CF patients produced significantly more proinflammatory cytokine than did control cells following exposure to the CF pathogens P. aeruginosa and Burkholderia cepacia complex (p < 0.001). Stimulation with pure TLR ligands demonstrated that TLR signaling appears to mediate the excessive cytokine production occurring in CF. Using complementary approaches involving both neutralizing Ab targeting TLR5 and flagellin-deficient bacteria, we established that inhibition of TLR5 abolished the damaging inflammatory response generated by CF airway cells following exposure to P. aeruginosa (p < 0.01). The potential therapeutic value of TLR5 inhibition was further supported by our demonstration that 75% of clinical isolates of P. aeruginosa retained TLR5 activating capacity during chronic CF lung infection. These studies identify the innate immune receptor TLR5 as a novel antiinflammatory target for reducing damaging lung inflammation in CF.
Noncytotoxic Functions of NK Cells: Direct Pathogen Restriction and Assistance to Adaptive Immunity
Authors: Strowig Till , Brilot Fabienne , Münz Christian
Published: April 2014

Natural killer cells were named after their ability to mediate spontaneous cytotoxicity during innate immune responses. However, it has become clear in recent years that they play an equally important role in restricting infections and assisting the development of adaptive immune responses via their ability to produce cytokines. In humans, a dedicated NK cell subset primarily fulfills these later functions. In this review we discuss the noncytotoxic effector functions of NK cells and how they could be harnessed for immunotherapy and vaccine development.
Spontaneous Development of a Pancreatic Exocrine Disease in CD28-Deficient NOD Mice
Authors: Meagher Craig , Tang Qizhi , Fife Brian T. , Bour-Jordan Helene , Wu Jenny , Pardoux Cecile , Bi Mingying , Melli Kristin , Bluestone Jeffrey A.
Published: April 2014

Autoimmune pancreatitis (AIP) is a heterogeneous autoimmune disease in humans characterized by a progressive lymphocytic and plasmacytic infiltrate in the exocrine pancreas. In this study, we report that regulatory T cell-deficient NOD.CD28KO mice spontaneously develop AIP that closely resembles the human disease. NOD mouse AIP was associated with severe periductal and parenchymal inflammation of the exocrine pancreas by CD4+ T cells, CD8+ T cells, and B cells. Spleen CD4+ T cells were found to be both necessary and sufficient for the development of AIP. Autoantibodies and autoreactive T cells from affected mice recognized a ∼50-kDa protein identified as pancreatic amylase. Importantly, administration of tolerogenic amylase-coupled fixed spleen cells significantly ameliorated disease severity, suggesting that this protein functions as a key autoantigen. The establishment and characterization of this spontaneous pancreatic amylase-specific AIP in regulatory T cell-deficient NOD.CD28KO mice provides an excellent model for the study of disease pathogenesis and development of new therapies for human autoimmune pancreatitis.
A Novel Trafficking Signal within the HLA-C Cytoplasmic Tail Allows Regulated Expression upon Differentiation of Macrophages
Authors: Schaefer Malinda R. , Williams Maya , Kulpa Deanna A. , Blakely Pennelope K. , Yaffee Anna Q. , Collins Kathleen L.
Published: April 2014

MHC class I molecules (MHC-I) present peptides to CTLs. In addition, HLA-C allotypes are recognized by killer cell Ig-like receptors (KIR) found on NK cells and effector CTLs. Compared with other classical MHC-I allotypes, HLA-C has low cell surface expression and an altered intracellular trafficking pattern. We present evidence that this results from effects of both the extracellular domain and the cytoplasmic tail. Notably, we demonstrate that the cytoplasmic tail contains a dihydrophobic (LI) internalization and lysosomal targeting signal that is partially attenuated by an aspartic acid residue (DXSLI). In addition, we provide evidence that this signal is specifically inhibited by hypophosphorylation of the adjacent serine residue upon macrophage differentiation and that this allows high HLA-C expression in this cell type. We propose that tightly regulated HLA-C surface expression facilitates immune surveillance and allows HLA-C to serve a specialized role in macrophages.
Pulmonary Surfactant Protein A Regulates TLR Expression and Activity in Human Macrophages
Authors: Henning Lisa N. , Azad Abul K. , Parsa Kishore V. L. , Crowther Joy E. , Tridandapani Susheela , Schlesinger Larry S.
Published: April 2014

The pulmonary innate immune system responds to various airborne microbes. Although its specificity is broad and based on the recognition of pathogen-associated molecular patterns, it is uniquely regulated to limit inflammation and thereby prevent damage to the gas-exchanging alveoli. Macrophages, critical cell determinants of this system, recognize microbes through pattern recognition receptors such as TLRs, which typically mediate proinflammatory responses. The lung collectin, surfactant protein A (SP-A), has emerged as an important innate immune determinant that regulates microbe-macrophage interactions in this environment. In this study, we report the basal and SP-A-induced transcriptional and posttranslational regulation of TLR2 and TLR4 expression during the differentiation of primary human monocytes into macrophages. Despite SP-A’s ability to up-regulate TLR2 expression on human macrophages, it dampens TLR2 and TLR4 signaling in these cells. SP-A decreases the phosphorylation of IκBα, a key regulator of NF-κB activity, and nuclear translocation of p65 which result in diminished TNF-α secretion in response to TLR ligands. SP-A also reduces the phosphorylation of TLR signaling proteins upstream of NF-κB, including members of the MAPK family. Finally, we report for the first time that SP-A decreases the phosphorylation of Akt, a major cell regulator of NF-κB and potentially MAPKs. These data identify a critical role for SP-A in modulating the lung inflammatory response by regulating macrophage TLR activity.
Lysine 144, a Ubiquitin Attachment Site in HIV-1 Nef, Is Required for Nef-Mediated CD4 Down-Regulation
Authors: Jin Yong-Jiu , Cai Catherine Yi , Zhang Xiaoping , Burakoff Steven J.
Published: April 2014

Nef is a HIV-1 accessory protein critical for the replication of the virus and the development of AIDS. The major pathological activity of Nef is the down-regulation of CD4, the primary receptor of HIV-1 infection. The mechanism underlying Nef-mediated CD4 endocytosis and degradation remains incompletely understood. Since protein ubiquitination is the predominant sorting signal in receptor endocytosis, we investigated whether Nef is ubiquitinated. The in vivo ubiquitination assay showed that both HIV-1 and SIV Nef proteins expressed in Jurkat T cells and 293T cells were multiple ubiquitinated by ubiquitin-His. The lysine-free HIV-1 Nef mutant (Δ10K) generated by replacing all 10 lysines with arginines was not ubiquitinated and the major ubiquitin-His attachment sites in HIV-1 Nef were determined to be lysine 144 (di-ubiquitinated) and lysine 204 (mono-ubiquitinated). Lysine-free HIV-1 Nef was completely inactive in Nef-mediated CD4 down-regulation, so was the Nef mutant with a single arginine substitution at K144 but not at K204. A mutant HIV-1 provirion NL4–3 with a single arginine substitution in Nef at K144 was also inactive in Nef-mediated CD4 down-regulation. Lysine-free Nef mutant reintroduced with lysine 144 (ΔK10 + K144) was shown active in CD4 down-regulation. These data suggest that ubiquitination of Nef, particularly diubiquitination of the lysine 144, is necessary for Nef-mediated CD4 down-regulation.
Trans-Presentation of IL-15 Dictates IFN-Producing Killer Dendritic Cells Effector Functions
Authors: Ullrich Evelyn , Bonmort Mathieu , Mignot Gregoire , Jacobs Benedikt , Bosisio Daniela , Sozzani Silvano , Jalil Abdelali , Louache Fawzia , Bulanova Elena , Geissman Frederic , Ryffel Bernard , Chaput Nathalie , Bulfone-Paus Silvia , Zitvogel Laurence
Published: April 2014

IFN-producing killer dendritic cells (IKDC) were initially described as B220+CD11c+CD3−NK1.1+ tumor-infiltrating cells that mediated part of the antitumor effects of the combination therapy with imatinib mesylate and IL-2. In this study, we show their functional dependency on IL-15 during homeostasis and inflammatory processes. Trans-presentation of IL-15 by IL-15Rα allows dramatic expansion of IKDC in vitro and in vivo, licenses IKDC for TRAIL-dependent killing and endows IKDC with immunizing potential, all three biological attributes not shared by B220−NK cells. However, IL-15 down-regulates the capacity of IKDC to induce MHC class I- or II-restricted T cell activation in vitro. Trans-presentation of IL-15 by IL-15Rα allows IKDC to respond to TLR3 and TLR4 ligands for the production of CCL2, a chemokine that is critical for IKDC trafficking into tumor beds (as described recently). We conclude that IKDC represent a unique subset of innate effectors functionally distinguishable from conventional NK cells in their ability to promptly respond to IL-15-driven inflammatory processes.
Inhibition of Dendritic Cell Maturation and Function Is Independent of Heme Oxygenase 1 but Requires the Activation of STAT3
Authors: Mashreghi Mir-Farzin , Klemz Roman , Knosalla Isabela Schmitt , Gerstmayer Bernhard , Janssen Uwe , Buelow Roland , Jozkowicz Alicja , Dulak Jozef , Volk Hans-Dieter , Kotsch Katja
Published: April 2014

The induction of heme oxygenase 1 (HO-1) by a single treatment with cobalt protoporphyrin (CoPPIX) protects against inflammatory liver failure and ischemia reperfusion injury after allotransplantation. In this context, the HO-1-mediated inhibition of donor-derived dendritic cell maturation and migration is discussed as one of the key events of graft protection. To investigate the poorly understood mechanism of CoPPIX-induced HO-1 activity in more detail, we performed gene expression analysis in murine liver, revealing the up-regulation of STAT3 after CoPPIX treatment. By using wild-type and HO-1-deficient dendritic cells we demonstrated that LPS-induced maturation is dependent on STAT3 phosphorylation and independent of HO-1 activity. In summary, our observations revise our understanding of the anti-inflammatory properties of HO-1 and highlight the immunomodulatory capacity of STAT3, which might be of further interest for targeting undesired immune responses, including ischemia reperfusion injury.
Regulation of IL-17 in Human CCR6+ Effector Memory T Cells
Authors: Liu Hong , Rohowsky-Kochan Christine
Published: April 2014

IL-17–secreting T cells represent a distinct CD4+ effector T cell lineage (Th17) that appears to be essential in the pathogenesis of numerous inflammatory and autoimmune diseases. Although extensively studied in the murine system, human Th17 cells have not been well characterized. In this study, we identify CD4+CD45RO+CCR7−CCR6+ effector memory T cells as the principal IL-17-secreting T cells. Human Th17 cells have a unique cytokine profile because the majority coexpress TNF-α but not IL-6 and a minor subset express IL-17 with IL-22 or IL-17 and IFN-γ. We demonstrate that the cytokines that promote the differentiation of human naive T cells into IL-17-secreting cells regulate IL-17 production by memory T cells. IL-1β alone or in association with IL-23 and IL-6 markedly increase IL-17+ CCR6+ memory T cells and induce IL-17 production in CCR6− memory T cells. We also show that T cell activation induces Foxp3 expression in T cells and that the balance between the percentage of Foxp3+ and IL-17+ T cells is inversely influenced by the cytokine environment. These studies suggest that the cytokine environment may play a critical role in the expansion of memory T cells in chronic autoimmune diseases.
IL-6, in Synergy with IL-7 or IL-15, Stimulates TCR-Independent Proliferation and Functional Differentiation of CD8+ T Lymphocytes
Authors: Gagnon Julien , Ramanathan Sheela , Leblanc Chantal , Cloutier Alexandre , McDonald Patrick P. , Ilangumaran Subburaj
Published: April 2014

Recent reports have shown that IL-21, in synergy with IL-15, stimulates proliferation of CD8+ T lymphocytes in the absence of signaling via the TCR. In this study, we show that IL-6, which induces phosphorylation of STAT3 similarly to IL-21, also can stimulate proliferation of CD8+ T cells in synergy with IL-7 or IL-15. IL-6 displays a stronger synergy with IL-7 than with IL-15 to stimulate naive CD8+ T cells. Concomitant stimulation by IL-6 or IL-21 augments phosphorylation and DNA-binding activity of STAT5 induced by IL-7 or IL-15. Like IL-21, IL-6 reduces the TCR signaling threshold required to stimulate CD8+ T cells. Prior culture of P14 TCR transgenic CD8 T cells with IL-6 or IL-21 in the presence of IL-7 or IL-15 augments their proliferation and cytolytic activity upon subsequent stimulation by Ag. Furthermore, cytokine stimulation induces quantitatively and qualitatively distinct phenotypic changes on CD8+ T cells compared with those induced by TCR signaling. We propose that the ability of IL-6 to induce TCR-independent activation of CD8+ T cells in synergy with IL-7 or IL-15 may play an important role in the transition from innate to adaptive immunity.
Retinoic Acid-Inducible Gene-I Mediates Late Phase Induction of TNF-α by Lipopolysaccharide
Authors: Wang Jing , Wu Su , Jin Xin , Li Mingtao , Chen Shiyong , Teeling Jessica L. , Perry V. Hugh , Gu Jun
Published: April 2014

LPS is the known component of bacterial pathogens that stimulates a number of proinflammatory factors. However, the mechanism of the induction of these factors by LPS has not been fully elucidated. We show here that LPS induces retinoic acid-inducible gene-I (RIG-I) in vitro and in vivo as a result from autocrine secretion of IFN-β in macrophages. TIR-domain-containing adapter-inducing IFN-β-deficient mouse embryo fibroblast (trif−/−) fail to show expression of RIG-I following LPS stimulation. Interference of RIG-I expression short interfering RNA represses the expression of LPS-induced TNF-α, whereas over-expression of RIG-I leads to the activation of TNF-α promoter and the induction of TNF-α expression. LPS- and IFN-β-induced TNF-α are suppressed in RIG-I-deficient mouse embryo fibroblasts (rig−/−). Thus, RIG-I plays a key role in the expression of TNF-α in macrophages in response to LPS stimulation, mainly for the late phase LPS-induced expression of TNF-α.
Proteasome Inhibitors Enhance TRAIL-Induced Apoptosis through the Intronic Regulation of DR5: Involvement of NF-κB and Reactive Oxygen Species-Mediated p53 Activation
Authors: Chen Jun-Jie , Chou Chia-Wei , Chang Yu-Fan , Chen Ching-Chow
Published: April 2014

Manipulation of TRAIL receptor 2 (DR5) pathway is a promising therapeutic strategy to overcome TRAIL-resistant lung cancer cells. Preclinical studies have shown that proteasome inhibitors enhance TRAIL-induced apoptosis in lung cancer cells, but the underlying mechanism has not been fully elucidated. In this study, we demonstrated the enhancement of TRAIL-mediated apoptosis in human alveolar epithelial cells by proteasome inhibitors that up-regulate DR5 expression. This effect was blocked by DR5-neutralizing Ab. Using reporter assay, we demonstrated that the p53 and NF-κB elements on the DR5 first intron region were involved in proteasome inhibitor-induced DR5 expression. Both p53 small interfering RNA and NF-κB inhibitor suppressed DR5 expression, strengthening the significance of p53 and NF-κB in DR5 transcription. The protein stability, Ser392 phosphorylation and Lys373/Lys382 acetylation of p53 were enhanced by MG132. In addition to p53, IκBα degradation and NF-κB translocation was also observed. Moreover, the binding of p53 and p65 to the first intron of DR5 was demonstrated by DNA affinity protein-binding and chromatin immunoprecipitation assays. Intracellular reactive oxygen species (ROS) generation after MG132 treatment contributed to p53, but not p65 nuclear translocation and DNA-binding activity. ROS scavenger dramatically inhibited the apoptosis induced by proteasome inhibitors plus TRAIL. The p53-null H1299 cells were resistant to proteasome inhibitor-induced DR5 up-regulation and enhancement of TRAIL-induced apoptosis. These findings reveal that proteasome inhibitor-mediated NF-κB and ROS-dependent p53 activation are contributed to intronic regulation of DR5 transcription, and resulted in the subsequent enhancement of TRAIL-induced apoptosis in human lung cancer cells.
ERK-Dependent Bim Modulation Downstream of the 4-1BB-TRAF1 Signaling Axis Is a Critical Mediator of CD8 T Cell Survival In Vivo
Authors: Sabbagh Laurent , Pulle Gayle , Liu Yuanqing , Tsitsikov Erdyni N. , Watts Tania H.
Published: April 2014

During an acute immune response, CD8 T cells undergo rapid expansion followed by a contraction phase during which the majority of activated T cells die, leaving a few survivors to persist as memory cells. The regulation of T cell survival is critical at each stage of this response. 4-1BB, a TNFR family member, has been implicated in prolonging the survival of activated and memory CD8 T cells; however, the precise mechanisms by which 4-1BB sustains T cell survival are incompletely understood. Upon aggregation on T cells, 4-1BB associates with two TNFR-associated factors (TRAF), TRAF1 and TRAF2. TRAF2 is essential for downstream signaling from 4-1BB; however, the role of TRAF1 in 4-1BB signaling has not been elucidated and there have been conflicting data as to whether TRAF1 provides a positive or a negative signal in T cells. In this study, we report that TRAF1 plays a critical role in survival signaling downstream of 4-1BB during CD8 T cell expansion in response to viral infection in vivo. Further analysis reveals that TRAF1-deficient cells are impaired in their ability to up-regulate the prosurvival Bcl-2 family member Bcl-xL and show increased levels of the proapoptotic Bcl-2 family member Bim following 4-1BB signaling. TRAF1-deficient CD8 T cells fail to activate ERK in response to 4-1BB ligation and inhibition of ERK signaling downstream of 4-1BB in wild-type cells leads to increased Bim levels. Thus, TRAF1 has a prosurvival effect in CD8 T cells via the 4-1BB-mediated up-regulation of Bcl-xL and ERK-dependent Bim down-modulation.
Histone Acetylation Facilitates Rapid and Robust Memory CD8 T Cell Response through Differential Expression of Effector Molecules (Eomesodermin and Its Targets: Perforin and Granzyme B)
Authors: Araki Yasuto , Fann Monchou , Wersto Robert , Weng Nan-ping
Published: April 2014

To understand the mechanism regulating the effector function of memory CD8 T cells, we examined expression and chromatin state of a key transcription factor (eomesodermin, EOMES) and two of its targets: perforin (PRF1) and granzyme B (GZMB). Accessible chromatin associated histone 3 lysine 9 acetylation (H3K9Ac) was found significantly higher at the proximal promoter and the first exon region of all three genes in memory CD8 T cells than in naive CD8 T cells. Correspondingly, EOMES and PRF1 were constitutively higher expressed in memory CD8 T cells than in naive CD8 T cells at resting and activated states. In contrast, higher expression of GZMB was induced in memory CD8 T cells than in naive CD8 T cells only after activation. Regardless of their constitutive or inducible expression, decreased H3K9Ac levels after treatment with a histone acetyltransferase inhibitor (Curcumin) led to decreased expression of all three genes in activated memory CD8 T cells. These findings suggest that H3K9Ac associated accessible chromatin state serves as a corner stone for the differentially high expression of these effector genes in memory CD8 T cells. Thus, epigenetic changes mediated via histone acetylation may provide a chromatin “memory” for the rapid and robust transcriptional response of memory CD8 T cells.
A Novel Adenovirus Expressing Flt3 Ligand Enhances Mucosal Immunity by Inducing Mature Nasopharyngeal-Associated Lymphoreticular Tissue Dendritic Cell Migration
Authors: Sekine Shinichi , Kataoka Kosuke , Fukuyama Yoshiko , Adachi Yasuo , Davydova Julia , Yamamoto Masato , Kobayashi Ryoki , Fujihashi Keiko , Suzuki Hideaki , Curiel David T. , Shizukuishi Satoshi , McGhee Jerry R. , Fujihashi Kohtaro
Published: April 2014

Previously, we showed that nasal administration of a naked cDNA plasmid expressing Flt3 ligand (FL) cDNA (pFL) enhanced CD4+ Th2-type, cytokine-mediated mucosal immunity and increased lymphoid-type dendritic cell (DC) numbers. In this study, we investigated whether targeting nasopharyngeal-associated lymphoreticular tissue (NALT) DCs by a different delivery mode of FL, i.e., an adenovirus (Ad) serotype 5 vector expressing FL (Ad-FL), would provide Ag-specific humoral and cell-mediated mucosal immunity. Nasal immunization of mice with OVA plus Ad-FL as mucosal adjuvant elicited high levels of OVA-specific Ab responses in external secretions and plasma as well as significant levels of OVA-specific CD4+ T cell proliferative responses and OVA-induced IFN-γ and IL-4 production in NALT, cervical lymph nodes, and spleen. We also observed higher levels of OVA-specific CTL responses in the spleen and cervical lymph nodes of mice given nasal OVA plus Ad-FL than in mice receiving OVA plus control Ad. Notably, the number of CD11b+CD11c+ DCs expressing high levels of costimulatory molecules was preferentially increased. These DCs migrated from the NALT to mucosal effector lymphoid tissues. Taken together, these results suggest that the use of Ad-FL as a nasal adjuvant preferentially induces mature-type NALT CD11b+CD11c+ DCs that migrate to effector sites for subsequent CD4+ Th1- and Th2-type cytokine-mediated, Ag-specific Ab and CTL responses.
CTLs Directed against HER2 Specifically Cross-React with HER3 and HER4
Authors: Conrad Heinke , Gebhard Kerstin , Krönig Holger , Neudorfer Julia , Busch Dirk H. , Peschel Christian , Bernhard Helga
Published: April 2014

The human epidermal growth factor receptor 2 (HER2) has been targeted as a breast cancer-associated Ag by T cell-based immunotherapeutical strategies such as cancer vaccines and adoptive T cell transfer. The prerequisite for a successful T cell-based therapy is the induction of T cells capable of recognizing the HER2-expressing tumor cells. In this study, we generated human cytotoxic T cell clones directed against the HER2369–377 epitope known to be naturally presented with HLA-A*0201. Those HER2-reactive CTLs, which were also tumor lytic, exhibited a similar lysis pattern dividing the targets in lysable and nonlysable tumor cells. Several HER2-expressing tumor cells became susceptible to CTL-mediated lysis after IFN-γ treatment and, in parallel, up-regulated molecules of the Ag-presenting machinery, indicating that the tumor itself also contributes to the success of CTL-mediated killing. Some of the HER2369–377-reactive T cells specifically cross-reacted with the corresponding peptides derived from the family members HER3 and/or HER4 due to a high sequence homology. The epitopes HER3356–364 and HER4361–369 were endogenously processed and contributed to the susceptibility of cell lysis by HER cross-reacting CTLs. The principle of “double” or “triple targeting” the HER Ags by cross-reacting T cells will impact the further development of T cell-based therapies.
Single-Chain TNF, a TNF Derivative with Enhanced Stability and Antitumoral Activity
Authors: Krippner-Heidenreich Anja , Grunwald Ingo , Zimmermann Gudrun , Kühnle Marie , Gerspach Jeannette , Sterns Theobald , Shnyder Steve D. , Gill Jason H. , Männel Daniela N. , Pfizenmaier Klaus , Scheurich Peter
Published: April 2014

The inflammatory and proapoptotic cytokine TNF possesses a compelling potential as an antitumoral therapeutic agent. Possible target cells include the malignant cells themselves, the tumor vasculature, or the immune system. As the clinical use of TNF is limited by systemic toxicity, targeting strategies using TNF-based fusion proteins are currently used. A major obstacle, however, is that homotrimeric TNF ligands are prone to activity loss due to dissociation into their monomers. In this study, we report the construction of single-chain TNF molecule, a TNF mutant consisting of three TNF monomers fused by short peptide linkers. In comparison to wild-type TNF, single-chain TNF was found to possess increased stability in vitro and in vivo, displayed reduced systemic toxicity yet slightly enhanced antitumoral activity in mouse models. Creation of single-chain variants is a new approach for improvement of functional activity of therapeutics based on TNF family ligands.