bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-4 Regulates MEK Expression Required for Lysophosphatidic Acid-Mediated Chemokine Generation by Human Mast Cells
Authors: Lin Debby A. , Boyce Joshua A.
Published: April 2014

IL-4 and mast cells (MCs) mediate mucosal defense against helminths and are central to allergic inflammation. Lysophosphatidic acid (LPA), an abundant, potent lipid growth factor, stimulates the growth of cultured human MCs (hMCs) in vitro through a pathway involving LPA receptors 1 and 3 (termed the LPA1 and LPA3 receptors, respectively) and peroxisome proliferator-activated receptor-γ. We now report that LPA potently induces the generation of proinflammatory chemokines (MIP-1β, IL-8, and MCP-1) by hMCs by a mechanism that absolutely requires IL-4. The de novo expression of chemokine mRNA and protein generation involves synergistic actions of calcium flux-dependent NFAT transcription factors and ERK. ERK phosphorylation and chemokine production in response to LPA require IL-4-dependent up-regulation of MEK-1 expression by a pathway involving PI3K. Although receptor-selective agonists for both the LPA2 and LPA3 receptors induce calcium fluxes by hMCs, only the LPA2 receptor-selective agonist fatty alcohol phosphate-12 mimics the IL-4-dependent effect of LPA on chemokine generation. The fact that LPA, an endogenous lipid mediator, activates hMCs by an LPA2 receptor-dependent pathway indicates functional distinctions between different LPA receptor family members that are expressed constitutively by cells of a single hemopoietic lineage. Moreover, the regulation of MEK-dependent signaling is a mechanism by which IL-4 could amplify inflammation in mucosal immune responses through receptor systems for endogenous ligands such as LPA.
Human NK Cytotoxicity against Porcine Cells Is Triggered by NKp44 and NKG2D
Authors: Forte Pietro , Lilienfeld Benjamin G. , Baumann Bettina C. , Seebach Jörg D.
Published: April 2014

Pig-to-human xenotransplantation has been proposed as a means to alleviate the shortage of human organs for transplantation, but cellular rejection remains a hurdle for successful xenograft survival. NK cells have been implicated in xenograft rejection and are tightly regulated by activating and inhibitory receptors recognizing ligands on potential target cells. The aim of the present study was to analyze the role of activating NK receptors including NKp30, NKp44, NKp46, and NKG2D in human xenogeneic NK cytotoxicity against porcine endothelial cells (pEC). 51Cr release and Ab blocking assays were performed using freshly isolated, IL-2-activated polyclonal NK cell populations as well as a panel of NK clones. Freshly isolated NK cells are NKp44 negative and lysed pEC exclusively in an NKG2D-dependent fashion. In contrast, the lysis of pEC mediated by activated human NK cells depended on both NKp44 and NKG2D, since a complete protection of pEC was achieved only by simultaneous blocking of these activating NK receptors. Using a panel of NK clones, a highly significant correlation between anti-pig NK cytotoxicity and NKp44 expression levels was revealed. Other triggering receptors such as NKp30 and NKp46 were not involved in xenogeneic NK cytotoxicity. Finally, Ab-dependent cell-mediated cytotoxicity of pEC mediated by human NK cells in the presence of xenoreactive Ab was not affected by blocking of activating NK receptors. In conclusion, strategies aimed to inhibit interactions between NKp44 and NKG2D on human NK cells and so far unknown ligands on pEC may prevent direct NK responses against xenografts but not xenogeneic Ab-dependent cell-mediated cytotoxicity.
The Control of Allergic Conjunctivitis by Suppressor of Cytokine Signaling (SOCS)3 and SOCS5 in a Murine Model
Authors: Ozaki Akemi , Seki Yoh-ichi , Fukushima Atsuki , Kubo Masato
Published: April 2014

Allergic conjunctivitis (AC) is a common allergic eye disease characterized by clinical symptoms such as itchiness, conjunctival congestion, elevated Ag-specific IgE, mast cell activation, and local eosinophil infiltration. In this study we established a murine model for Ag-induced AC to understand the pathogenesis of the disease. Cell transfer experiments indicated that AC can be divided into early and late phase responses (EPR and LPR). EPR was associated with IgE responses, leading to itchiness, whereas LPR was characterized by local eosinophil infiltration. Both EPR and LPR were significantly inhibited in STAT6-deficient mice, and adoptive transfer of Th2 cells reconstituted LPR. Furthermore, SOCS3 was highly expressed at the disease site, and T cell-specific expression of SOCS3 deteriorated clinical and pathological features of AC, indicating that Th2-mediated SOCS3 expression controls the development and persistence of AC. Reduction of the expression level in SOCS3 heterozygous mice or inhibition of function in dominant-negative SOCS3 transgenic mice clearly reduced the severity of AC. In contrast, constitutive expression of SOCS5, a specific inhibitor of IL-4 signaling, resulted in reduced eosinophil infiltration. These results suggest that negative regulation of the Th2-mediated response by dominant-negative SOCS3 and SOCS5 could be a target for therapeutic intervention in allergic disease.
Treatment with a Laminin-Derived Peptide Suppresses Lupus Nephritis
Authors: Amital Howard , Heilweil Michal , Ulmansky Rina , Szafer Fanny , Bar-Tana Ruth , Morel Laurence , Foster Mary H. , Mostoslavsky Gustavo , Eilat Dan , Pizov Galina , Naparstek Yaakov
Published: April 2014

The role of DNA as the target for pathogenic lupus autoantibodies in systemic lupus erythematosus is equivocal and renal damage may be due to cross-reactivity of lupus Abs with glomerular components. We have previously shown that lupus autoantibodies bind to the laminin component of the extracellular matrix. In the present work, we have analyzed the fine specificity of the interaction of pathogenic murine lupus autoantibodies with this molecule and the effect of inhibiting their binding to laminin during the course of the disease. We have found that pathogenic murine lupus autoantibodies react with a 21-mer peptide located in the globular part of the α-chain of laminin. Immunization of young lupus-prone mice with this peptide accelerated renal disease. Analysis of transgenic, congenic, and RAG-1−/− mice confirmed the importance of this epitope in the pathogenesis of lupus renal disease. We have synthesized a panel of peptides that cross-react with the anti-laminin Abs and have found that the binding of lupus autoantibodies to the extracellular matrix could be inhibited in vitro by some of these competitive peptides. Treatment of MRL/lpr/lpr mice with these peptides prevented Ab deposition in the kidneys, ameliorated renal disease, and prolonged survival of the peptide-treated mice. We suggest that laminin components can serve as the target for lupus Abs. The interaction with these Ags can explain both the tissue distribution and the immunopathological findings in lupus. Moreover, inhibition of autoantibody binding to the extracellular matrix can lead to suppression of disease.
Inhibition of NK Cell Activity through TGF-β1 by Down-Regulation of NKG2D in a Murine Model of Head and Neck Cancer
Authors: Dasgupta Santanu , Bhattacharya-Chatterjee Malaya , O’Malley Bert W. , Chatterjee Sunil K.
Published: April 2014

In an orthotopic murine model of head and neck squamous cell carcinoma (SCC VII/SF) we studied NK cell-mediated immunity following vaccination with a recombinant vaccinia virus expressing IL-2 (rvv-IL-2). SCC VII/SF tumor cells were injected into the oral cavity of C3H/HeJ mice on day 0. Mice were vaccinated on days 7, 10, and 14 with rvv-IL-2 and control vaccines. Phenotypes, numbers, and biological activities of NK cells were determined following vaccination. Levels of expression of NK-activating receptor NKG2D and CD16 on NK cell surface were assayed in the vaccinated mice. Expression of NKG2D ligands, Rae1, and H60 on SCC VII/SF cells was also examined. Vaccination with rvv-IL-2 resulted in expansion of NK cells. NK cells isolated from rvv-IL-2-vaccinated mice had significantly higher biological activities compared with mice treated with control vaccines. NK cells from tumor-bearing mice expressed significantly lower levels of NKG2D and CD16 compared with rvv-IL-2 vaccinated mice. SCC VII/SF tumors expressed NKG2D ligand Rae 1, although H60 was not present. SCC VII/SF tumors expressed high levels of TGF-β1, which were down-modulated by vaccination with rvv-IL-2. Incubation of NK cells with tumor homogenate or cultured supernatant of SCC VII/SF cells reduced the expression of NKG2D and CD16. This inhibition appeared to be mediated by TGF-β1. SCC VII/SF tumors in the oral cavity of the mice secrete high quantities of TGF-β1, which reduce the expression of NK cell receptor NKG2D as well as CD16 and inhibits biological functions of NK cells.
Cutting Edge: Cytotoxic Granule Polarization and Cytolysis Can Occur without Central Supramolecular Activation Cluster Formation in CD8+ Effector T Cells
Authors: O’Keefe James P. , Gajewski Thomas F.
Published: April 2014

The functional implication of molecular segregation within the immunological synapse remains uncertain. We recently reported that effector but not naive TCR transgenic murine CD8+ T cells formed immunological synapses containing a central supramolecular activation cluster (cSMAC), suggesting that execution of effector functions such as cytolytic activity might be facilitated by the cSMAC structure. We have now explored this hypothesis using two approaches. First, by simultaneously imaging cSMAC formation and mobilization of cytotoxic granules to the synapse, we observed no correlation between the presence of a cSMAC and granule reorientation. Second, we took advantage of the observation that CD28 costimulation markedly enhances cSMAC formation. Granule polarization to the contact site was indistinguishable with B7-1+ and B7-1− target cells, and cytolytic activity against B7-1+ or B7-1− targets was similar and granule-dependent. Together, our results indicate that the formation of a cSMAC is not required for cytolytic activity in CD8+ effector T cells.
Cholera Toxin B-Subunit Prevents Activation and Proliferation of Human CD4+ T Cells by Activation of a Neutral Sphingomyelinase in Lipid Rafts
Authors: Rouquette-Jazdanian Alexandre K. , Foussat Arnaud , Lamy Laurence , Pelassy Claudette , Lagadec Patricia , Breittmayer Jean-Philippe , Aussel Claude
Published: April 2014

The inhibition of human CD4+ T lymphocyte activation and proliferation by cholera toxin B-subunit (CTB) is a well-established phenomenon; nevertheless, the exact mechanism remained unclear. In the present study, we propose an explanation for the rCTB-induced inhibition of CD4+ T lymphocytes. rCTB specifically binds to GM1, a raft marker, and strongly modifies the lipid composition of rafts. First, rCTB inhibits sphingomyelin synthesis; second, it enhances phosphatidylcholine synthesis; and third, it activates a raft-resident neutral sphingomyelinase resembling to neutral sphingomyelinase type 1, thus generating a transient ceramide production. We demonstrated that these ceramides inhibit protein kinase Cα phosphorylation and its translocation into the modified lipid rafts. Furthermore, we show that rCTB-induced ceramide production activate NF-κB. Combined all together: raft modification in terms of lipids, ceramide production, protein kinase Cα inhibition, and NF-κB activation lead to CD4+ T cell inhibition.
Durable Human Memory T Cells Quantifiable by Cultured Enzyme-Linked Immunospot Assays Are Induced by Heterologous Prime Boost Immunization and Correlate with Protection against Malaria
Authors: Keating Sheila M. , Bejon Philip , Berthoud Tamara , Vuola Jenni M. , Todryk Stephen , Webster Daniel P. , Dunachie Susanna J. , Moorthy Vasee S. , McConkey Samuel J. , Gilbert Sarah C. , Hill Adrian V. S.
Published: April 2014

Immunological memory is a required component of protective antimalarial responses raised by T cell-inducing vaccines. The magnitude of ex vivo IFN-γ T cell responses is widely used to identify immunogenic vaccines although this response usually wanes and may disappear within weeks. However, protection in the field is likely to depend on durable central memory T cells that are not detected by this assay. To identify longer-lived memory T cells, PBMC from malaria-naive vaccinated volunteers who had received prime boost vaccinations with a combination of DNA and/or viral vectors encoding the multiepitope string-thrombospondin-related adhesion protein Ag were cultured in vitro with Ag for 10 days before the ELISPOT assay. Ex vivo T cell responses peaked at 7 days after the final immunization and declined substantially over 6 mo, but responses identified after T cell culture increased over the 6-mo period after the final immunization. Moreover, individual cultured ELISPOT responses at the day of challenge time point correlated significantly with degree of protection against malaria sporozoite challenge, whereas ex vivo responses did not, despite a correlation between the peak ex vivo response and magnitude of memory responses 6 mo later. This cultured assay identifies long-lasting protective T cell responses and therefore offers an attractive option for assessments of vaccine immunogenicity.
Regulation of B7.1 Costimulatory Molecule Is Mediated by the IFN Regulatory Factor-7 through the Activation of JNK in Lipopolysaccharide-Stimulated Human Monocytic Cells
Authors: Lim Wilfred , Gee Katrina , Mishra Sasmita , Kumar Ashok
Published: April 2014

The engagement of CD28 or CTLA-4 with B7.1 provides the essential second costimulatory signal that regulates the development of immune responses, including T cell activation, differentiation, and induction of peripheral tolerance. The signaling molecules and the transcription factors involved in B7.1 regulation are poorly understood. In this study we investigated the role of MAPKs in the regulation of LPS-induced B7.1 expression in human monocytes and the promonocytic THP-1 cells. Our results show that LPS-induced B7.1 expression in monocytic cells did not involve the activation of either p38 or ERKs. Using the JNK-specific inhibitor SP600125, small interfering RNAs specific for JNK1 and JNK2, and agents such as dexamethasone that inhibit JNK activation, we determined that LPS-induced B7.1 expression was regulated by JNK MAPK in both monocytes and THP-1 cells. In addition, we identified a distinct B7.1-responsive element corresponding to the IFN regulatory factor-7 (IRF-7) binding site in the B7.1 promoter responsible for the regulation of LPS-induced B7.1 transcription. Furthermore, SP600125 and dexamethasone inhibited LPS-induced IRF-7 activity. Taken together, these results suggest that LPS-induced B7.1 transcription in human monocytic cells may be regulated by JNK-mediated activation of the IRF-7 transcription factor.
Induction of Uteroglobin-Related Protein 2 (Ugrp2) Gene Expression by the Th2 Cytokines IL-4 and IL-13
Authors: Yamada Atsushi , Sheikh Faruk , Niimi Tomoaki , DeMayo Francesco J. , Keegan Achsah D. , Donnelly Raymond P. , Kimura Shioko
Published: April 2014

Uteroglobin-related proteins 1 and 2 (UGRP1 and -2) are thought to play important roles in inflammation and immunologic responses in the lung. In this study we demonstrate that IL-4 and IL-13 enhance Ugrp2 gene expression in the mouse transformed Clara cell line, mtCC, in a time- and dose-dependent manner. Addition of actinomycin D abrogated the IL-4- and IL-13-induced increase of Ugrp2 expression, demonstrating that this increase occurs at the transcriptional level. When mtCC cells were pretreated with IFN-γ before the addition of IL-4 or IL-13, IL-4- and 13-induced Ugrp2 mRNA increase was markedly decreased. IL-4 and IL-13 induced phosphorylation of STAT6 in mtCC cells, which binds to the proximal STAT-binding element (SBE) in the Ugrp2 gene promoter, leading to transcriptional activation of this gene. Mutations of the proximal SBE abrogated the binding of activated STAT6 to this site and the IL-4-induced increase in Ugrp2 gene promoter activity. IFN-γ-activated STAT1 binds to the same SBE in the Ugrp2 gene promoter to which STAT6 binds and decreases the binding of STAT6 to this site. Furthermore, an IL-4-induced increase in Ugrp2 expression was not observed in primary cultures of lung cells derived from STAT6-deficient mice. These results indicate that Ugrp2 expression is enhanced by IL-4 and IL-13 through STAT6 binding to the proximal SBE located in the Ugrp2 gene promoter.
Ex Vivo Rapamycin Generates Donor Th2 Cells That Potently Inhibit Graft-versus-Host Disease and Graft-versus-Tumor Effects via an IL-4-Dependent Mechanism
Authors: Foley Jason E. , Jung Unsu , Miera Angel , Borenstein Todd , Mariotti Jacopo , Eckhaus Michael , Bierer Barbara E. , Fowler Daniel H.
Published: April 2014

Rapamycin (sirolimus) inhibits graft-vs-host disease (GVHD) and polarizes T cells toward Th2 cytokine secretion after allogeneic bone marrow transplantation (BMT). Therefore, we reasoned that ex vivo rapamycin might enhance the generation of donor Th2 cells capable of preventing GVHD after fully MHC-disparate murine BMT. Using anti-CD3 and anti-CD28 costimulation, CD4+ Th2 cell expansion was preserved partially in high-dose rapamycin (10 μM; Th2.rapa cells). Th2.rapa cells secreted IL-4 yet had reduced IL-5, IL-10, and IL-13 secretion relative to control Th2 cells. BMT cohorts receiving wild-type (WT) Th2.rapa cells, but not Th2.rapa cells generated from IL-4-deficient (knockout) donors, had marked Th2 skewing post-BMT and greatly reduced donor anti-host T cell alloreactivity. Histologic studies demonstrated that Th2.rapa cell recipients had near complete abrogation of skin, liver, and gut GVHD. Overall survival in recipients of WT Th2.rapa cells, but not IL-4 knockout Th2.rapa cells, was constrained due to marked attenuation of an allogeneic graft-vs-tumor (GVT) effect against host-type breast cancer cells. Delay in Th2.rapa cell administration until day 4, 7, or 14 post-BMT enhanced GVT effects, moderated GVHD, and improved overall survival. Therefore, ex vivo rapamycin generates enhanced donor Th2 cells for attempts to balance GVHD and GVT effects.
Physiological Levels of 15-Deoxy-Δ12,14-Prostaglandin J2 Prime Eotaxin-Induced Chemotaxis on Human Eosinophils through Peroxisome Proliferator-Activated Receptor-γ Ligation
Authors: Kobayashi Yoshiki , Ueki Shigeharu , Mahemuti Gulixian , Chiba Takahito , Oyamada Hajime , Saito Norihiro , Kanda Akira , Kayaba Hiroyuki , Chihara Junichi
Published: April 2014

15-Deoxy-Δ12,14-PGJ2 (15d-PGJ2), mainly produced by mast cells, is known as a potent lipid mediator derived from PGD2 in vivo. 15d-PGJ2 was thought to exert its effects on cells exclusively through peroxisome proliferator-activated receptor-γ (PPARγ) and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), which are both expressed on human eosinophils. However, the physiological role of 15d-PGJ2 remains unclear, because the concentration generated in vivo is generally much lower than that required for its biological functions. In the present study we found that low concentrations (picomolar to low nanomolar) of 15d-PGJ2 and a synthetic PPARγ agonist markedly enhanced the eosinophil chemotaxis toward eotaxin, and the effect was decreased in a dose-dependent manner. Moreover, at a low concentration (10−10 M), 15d-PGJ2 and troglitazone primed eotaxin-induced shape change and actin polymerization. These priming effects were completely reversed by a specific PPARγ antagonist, but were not mimicked by CRTH2 agonist 13,14-dihydro-15-keto-PGD2, suggesting that the effects were mediated through PPARγ ligation. The effect exerted by 15d-PGJ2 parallels the enhancement of Ca2+ influx, but is not associated with the ERK, p38 MAPK, and NF-κB pathways. Furthermore, the time course and treatment of eosinophils with actinomycin D, an inhibitor of gene transcription, indicated that the transcription-independent pathway had a role in this process. PPARγ might interact with an eotaxin-induced cytosolic signaling pathway, because PPARγ is located in the eosinophil cytosol. Taken together with current findings, these results suggest that under physiological conditions, 15d-PGJ2 contributes to allergic inflammation through PPARγ, which plays a role as a biphasic regulator of immune response.
Human CD4+ T Cells Are Predominantly Distributed among Six Phenotypically and Functionally Distinct Subsets
Authors: Amyes Elisabeth , McMichael Andrew J. , Callan Margaret F. C.
Published: April 2014

Human T cells are heterogeneous, varying in terms of their phenotype, functional capabilities, and history of Ag encounter. The derivation of a functionally relevant model for classifying CD4+ T cells has been hampered by limitations on the numbers of parameters that may be measured using classical four-color flow cytometry. In this study we have taken advantage of the introduction of reagents for five-color flow cytometry to develop a detailed, functionally meaningful scheme for classifying human CD4+ T cells. We show that CD4+ T cells are predominantly distributed among six of eight possible compartments, identified by the expression of CCR7, CD45RA, and CD28. We demonstrate novel phenotypic and functional correlates that justify the choice of these three molecules to define CD4+ T cell compartments. We note that CD4+ T cells with different Ag specificities are distributed differently among the six described subsets. On the basis of these results, we propose a cross-sectional model for classification of peripheral CD4+ T cells. Knowledge of where T cells lie on this model informs about their functional capacity and can reflect their history of Ag exposure.
Differential Effects of B and T Lymphocyte Attenuator and Programmed Death-1 on Acceptance of Partially versus Fully MHC-Mismatched Cardiac Allografts
Authors: Tao Ran , Wang Liqing , Han Rongxiang , Wang Tao , Ye Qunrui , Honjo Takasu , Murphy Theresa L. , Murphy Kenneth M. , Hancock Wayne W.
Published: April 2014

Although fully MHC-mismatched murine cardiac allografts are rapidly rejected, allografts mismatched at only MHC class I or class II alleles survive long term; the immunologic basis for the long-term survival of MHC class I- or II-mismatched allografts is unknown. We examined the roles of two recently described inhibitory receptors, B and T lymphocyte attenuator (BTLA) and programmed death-1 (PD-1), in the survival of partially or fully MHC-mismatched allografts using gene-deficient recipients as well as through use of blocking mAbs in wild-type hosts. Partially MHC-mismatched allografts showed strong induction of BTLA, but not PD-1 mRNA and survived long term in wild-type recipients, whereas targeting of BTLA or its ligand, herpesvirus entry mediator, but not PD-1, prompted their rapid rejection. By contrast, fully MHC-mismatched cardiac allografts were acutely rejected in wild-type recipients despite the induction of both BTLA and PD-1. Targeting of PD-1 in several fully MHC-mismatched models accelerated rejection, whereas targeting of BTLA unexpectedly enhanced PD-1 induction by alloreactive CD4 and CD8 T cells and prolonged allograft survival. In vitro studies using allogeneic dendritic cells and T cells showed that at low levels of T cell activation, BTLA expression was primarily induced, but that with increasing degrees of T cell activation, the expression of PD-1 was strongly up-regulated. These data suggest that BTLA and PD-1 exert distinct inhibitory actions in vivo, with the BTLA/herpesvirus entry mediator pathway appearing to dominate in regulating responses against a restricted degree of allogeneic mismatch.
NK Cells Infiltrating a MHC Class I-Deficient Lung Adenocarcinoma Display Impaired Cytotoxic Activity toward Autologous Tumor Cells Associated with Altered NK Cell-Triggering Receptors
Authors: Le Maux Chansac Béatrice , Moretta Alessandro , Vergnon Isabelle , Opolon Paule , Lécluse Yann , Grunenwald Dominique , Kubin Marek , Soria Jean-Charles , Chouaib Salem , Mami-Chouaib Fathia
Published: April 2014

NK cells are able to discriminate between normal cells and cells that have lost MHC class I (MHC-I) molecule expression as a result of tumor transformation. This function is the outcome of the capacity of inhibitory NK receptors to block cytotoxicity upon interaction with their MHC-I ligands expressed on target cells. To investigate the role of human NK cells and their various receptors in the control of MHC-I-deficient tumors, we have isolated several NK cell clones from lymphocytes infiltrating an adenocarcinoma lacking β2-microglobulin expression. Unexpectedly, although these clones expressed NKG2D and mediated a strong cytolytic activity toward K562, Daudi and allogeneic MHC-class I+ carcinoma cells, they were unable to lyse the autologous MHC-I− tumor cell line. This defect was associated with alterations in the expression of natural cytotoxicity receptor (NCR) by NK cells and the NKG2D ligands, MHC-I-related chain A, MHC-I-related chain B, and UL16 binding protein 1, and the ICAM-1 by tumor cells. In contrast, the carcinoma cell line was partially sensitive to allogeneic healthy donor NK cells expressing high levels of NCR. Indeed, this lysis was inhibited by anti-NCR and anti-NKG2D mAbs, suggesting that both receptors are required for the induced killing. The present study indicates that the MHC-I-deficient lung adenocarcinoma had developed mechanisms of escape from the innate immune response based on down-regulation of NCR and ligands required for target cell recognition.
ES-62, an Immunomodulator Secreted by Filarial Nematodes, Suppresses Clonal Expansion and Modifies Effector Function of Heterologous Antigen-Specific T Cells In Vivo
Authors: Marshall Fraser A. , Grierson Angela M. , Garside Paul , Harnett William , Harnett Margaret M.
Published: April 2014

ES-62 is a phosphorylcholine-containing glycoprotein secreted by filarial nematodes, which has previously been shown to possess a range of immunomodulatory capabilities. We now show, using a CD4+ transgenic TCR T cell adoptive transfer system, that ES-62 can modulate heterologous Ag (OVA)-specific responses in vivo. Thus, in contrast to the mixed IgG1-IgG2a response observed in control animals, ES-62-treated mice exhibited a Th2-biased IgG Ab response as evidenced by stable enhancement of anti-OVA IgG1 production and a profound inhibition of anti-OVA IgG2a. Consistent with this, Ag-specific IFN-γ produced was suppressed by pre-exposure to ES-62 when T cells were rechallenged ex vivo. However, the response observed was not classical Th2, because although Ag-specific IL-5 production was enhanced by pre-exposure to ES-62, IL-13, and IL-4 were inhibited when T cells were rechallenged ex vivo. Moreover, such T cells produced lower levels of IL-2 and proliferated less upon Ag rechallenge ex vivo. Finally, pre-exposure to ES-62 inhibited the clonal expansion of the transferred Ag-specific CD4+ T cells and altered the functional response of such T cells in vivo, by modulating the kinetics and reducing the extent of their migration into B cell follicles.
The Earliest Thymic Progenitors in Adults Are Restricted to T, NK, and Dendritic Cell Lineage and Have a Potential to Form More Diverse TCRβ Chains than Fetal Progenitors
Authors: Lu Min , Tayu Risa , Ikawa Tomokatsu , Masuda Kyoko , Matsumoto Isamu , Mugishima Hideo , Kawamoto Hiroshi , Katsura Yoshimoto
Published: April 2014

T cell progenitors in the adult thymus (AT) are not well characterized. In the present study, we show that the earliest progenitors in the murine AT are, like those in fetal thymus (FT), unable to generate B or myeloid cells, but still retain the ability to generate NK cells and dendritic cells. However, AT progenitors are distinct from those in FT or fetal liver, in that they are able to produce ∼100 times larger numbers of T cells than progenitors in fetuses. Such a capability to generate a large number of T cells was mainly attributed to their potential to extensively proliferate before the TCRβ chain gene rearrangement. We propose that the AT is colonized by T/NK/dendritic cell tripotential progenitors with much higher potential to form diversity in TCRβ chains than FT progenitors.
IL-12 Breaks Dinitrothiocyanobenzene (DNTB)-Mediated Tolerance and Converts the Tolerogen DNTB into an Immunogen
Authors: Riemann Helge , Loser Karin , Beissert Stefan , Fujita Mayumi , Schwarz Agatha , Schwarz Thomas , Grabbe Stephan
Published: April 2014

Epicutaneous application of dinitrothiocyanobenzene (DNTB) induces tolerance against its related compound dinitrofluorobenzene (DNFB), because DNTB-pretreated mice cannot be sensitized against the potent hapten DNFB. This tolerance is hapten-specific and transferable. In this study, we demonstrate that IL-12 can break DNTB-mediated tolerance. Furthermore, naive mice treated with IL-12 before DNTB application responded to DNFB challenge with a pronounced ear swelling response without previous sensitization to DNFB, showing that IL-12 can convert the tolerogen DNTB into an immunogen. No differences in numbers or regulatory activity were observed between CD4+CD25+ regulatory T cells isolated from mice treated with DNFB, DNTB, or IL-12 followed by DNTB. However, the number of CD207+ Langerhans cells in regional lymph nodes of DNTB-treated mice was significantly lower than in animals treated with DNFB or IL-12 plus DNTB. Additionally, CD11c+ dendritic cells (DC) isolated from regional lymph nodes of DNTB-treated mice had a significantly lower ability to stimulate T cell proliferation and produced reduced amounts of inflammatory cytokines. Application of both DNFB and DNTB induced apoptotic cell death of DC in the epidermis and the regional lymph nodes. However, the number of apoptotic DC in regional lymph nodes was significantly higher in DNTB-treated animals compared with mice treated with DNFB or IL-12 plus DNTB. Therefore, we conclude that DNTB-mediated tolerance is secondary to inefficient Ag presentation as a result of apoptotic cell death of DC and that IL-12 converts the tolerogen DNTB into an immunogen by preventing DNTB-induced apoptosis of DC.
IFN-γ Production by CD8+ T Cells Depends on NFAT1 Transcription Factor and Regulates Th Differentiation
Authors: Teixeira Leonardo K. , Fonseca Bruna P. F. , Vieira-de-Abreu Adriana , Barboza Bianca A. , Robbs Bruno K. , Bozza Patrícia T. , Viola João P. B.
Published: April 2014

CD8+ T lymphocytes are excellent sources of IFN-γ; however, the molecular mechanisms that dictate IFN-γ expression upon TCR stimulation in these cells are not completely understood. In this study, we evaluated the involvement of NFAT1 in the regulation of IFN-γ gene expression in murine CD8+ T cells and its relevance during Th differentiation. We show that CD8+, but not CD4+, T cells, represent the very first source of IFN-γ upon primary T cell activation, and also that the IFN-γ produced by naive CD8+ T cells may enhance CD4+ Th1 differentiation in vitro. TCR stimulation rapidly induced IFN-γ expression in CD8+ T lymphocytes in a cyclosporin A-sensitive manner. Evaluation of CD8+ T cells showed that calcium influx alone was sufficient to activate NFAT1 protein, transactivate IFN-γ gene promoter, and induce IFN-γ production. In fact, NFAT1-deficient mice demonstrated highly impaired IFN-γ production by naive CD8+ T lymphocytes, which were totally rescued after retroviral transduction with NFAT1-encoding vectors. Moreover, NFAT1-dependent IFN-γ production by the CD8+ T cell compartment was crucial to control a Th2-related response in vivo, such as allergic inflammation. Consistently, CD8α- as well as IFN-γ-deficient mice did not mount a Th1 immune response and also developed in vivo allergic inflammation. Our results clearly indicate that IFN-γ production by CD8+ T cells is dependent of NFAT1 transcription factor and may be an essential regulator of Th immune responses in vivo.
Differential Processing of Autoantigens in Lysosomes from Human Monocyte-Derived and Peripheral Blood Dendritic Cells
Authors: Burster Timo , Beck Alexander , Tolosa Eva , Schnorrer Petra , Weissert Robert , Reich Michael , Kraus Marianne , Kalbacher Hubert , Häring Hans-Ulrich , Weber Ekkehard , Overkleeft Herman , Driessen Christoph
Published: April 2014

Dendritic cells (DC) initiate immunity and maintain tolerance. Although in vitro-generated DC, usually derived from peripheral blood monocytes (MO-DC), serve as prototype DC to analyze the biology and biochemistry of DC, phenotypically distinct primary types of DC, including CD1c-DC, are present in peripheral blood (PB-DC). The composition of lysosomal proteases in PB-DC and the way their MHC class II-associated Ag-processing machinery handles a clinically relevant Ag are unknown. We show that CD1c-DC lack significant amounts of active cathepsins (Cat) S, L, and B as well as the asparagine-specific endopeptidase, the major enzymes believed to mediate MHC class II-associated Ag processing. However, at a functional level, lysosomal extracts from CD1c-DC processed the multiple sclerosis-associated autoantigens myelin basic protein and myelin oligodendrocyte glycoprotein in vitro more effectively than MO-DC. Although processing was dominated by CatS, CatD, and asparagine-specific endopeptidase in MO-DC, it was dominated by CatG in CD1c-DC. Thus, human MO-DC and PB-DC significantly differ with respect to their repertoire of active endocytic proteases, so that both proteolytic machineries process a given autoantigen via different proteolytic pathways
Impact of Peptides on the Recognition of HLA Class I Molecules by Human HLA Antibodies
Authors: Mulder Arend , Eijsink Chantal , Kester Michel G. D. , Franke Marry E. I. , Kardol Marrie J. , Heemskerk Mirjam H. M. , van Kooten Cees , Verreck Frank A. , Drijfhout Jan Wouter , Koning Frits , Doxiadis Ilias I. N. , Claas Frans H. J.
Published: April 2014

MHC class I molecules expressed on cell surfaces are composed of H chain, β2-microglobulin and any of a vast array of peptides. The role of peptide in the recognition of HLA class I by serum HLA Abs is unknown. In this study, the solid-phase assay of a series (n = 11) of HLA-A2-reactive, pregnancy-induced, human mAbs on a panel (n = 12) of recombinant monomeric HLA-A2 molecules, each containing a single peptide, revealed peptide selectivity of the mAbs. The flow cytometry membrane staining intensities on the HLA-A2-transduced cell line K562, caused by these mAbs, correlated with the number of monomer species detected by the mAbs. Flow cytometry staining on HLA-A2-bearing cell lines of a variety of lineages was indicative of tissue selectivity of these HLA-A2 mAbs. This tissue selectivity suggests that the deleterious effect on allografts is confined to alloantibodies recognizing only HLA class I loaded with peptides that are derived from tissue-specific and household proteins. Since Abs that are only reactive with HLA loaded with irrelevant peptides are expected to be harmless toward allografts, the practice of HLA Ab determination on lymphocyte-derived HLA deserves reconsideration.
MARCO Is the Major Binding Receptor for Unopsonized Particles and Bacteria on Human Alveolar Macrophages
Authors: Arredouani Mohamed S. , Palecanda Aiyappa , Koziel Henry , Huang Yuh-Ching , Imrich Amy , Sulahian Timothy H. , Ning Yao Yu , Yang Zhiping , Pikkarainen Timo , Sankala Marko , Vargas Sara O. , Takeya Motohiro , Tryggvason Karl , Kobzik Lester
Published: April 2014

Alveolar macrophages (AMs) avidly bind and ingest inhaled environmental particles and bacteria. To identify the particle binding receptor(s) on human AMs, we used functional screening of anti-human AM hybridomas and isolated a mAb, PLK-1, which inhibits AM binding of unopsonized particles (e.g., TiO2, latex beads; 63 ± 5 and 67 ± 4% inhibition, respectively, measured by flow cytometry; n = 11) and unopsonized bacteria (∼84 and 41% inhibition of Escherichia coli and Staphylococcus aureus binding by mAb PLK-1, respectively). The PLK-1 Ag was identified as the human class A scavenger receptor (SR) MARCO (macrophage receptor with collagenous structure) by observing specific immunolabeling of COS cells transfected with human MARCO (but not SR-AI/II) cDNA and by immunoprecipitation by PLK-1 of a protein of appropriate molecular mass (∼70 kDa) from both normal human bronchoalveolar lavage cells (>90% AMs) and human MARCO-transfected COS cells. PLK-1 also specifically inhibited particle binding by COS cells, only after transfection with human MARCO cDNA. Immunostaining showed specific labeling of AMs within human lung tissue, bronchoalveolar lavage samples, as well as macrophages in other sites (e.g., lymph node and liver). Using COS transfectants with different truncated forms of MARCO, allowed epitope mapping for the PLK-1 Ab to MARCO domain V between amino acid residues 420 and 431. A panel of Abs to various SRs identified expression on AMs, but failed to inhibit TiO2 or S. aureus binding. The data support a dominant role for MARCO in the human AM defense against inhaled particles and pathogens.
Low-Level CD4+ T Cell Activation Is Associated with Low Susceptibility to HIV-1 Infection
Authors: Koning Fransje A. , Otto Sigrid A. , Hazenberg Mette D. , Dekker Linda , Prins Maria , Miedema Frank , Schuitemaker Hanneke
Published: April 2014

Different features have been associated with low susceptibility to HIV type 1 (HIV-1) infection in exposed seronegative individuals. These include genetic make-up such as homozygosity for the CCR5-Δ32 allele and the presence of HIV-specific CTLs. We studied immune activation and immune responsiveness in relation to HIV-1 susceptibility in 42 high-risk seronegative (HRSN) participants of the Amsterdam Cohort Studies and 54 men from the same cohort who were seronegative at the moment of analysis but later became HIV seropositive. HRSN had higher naive (CD45RO CD27) CD4 and CD8 T cell numbers and lower percentages of activated (HLADR CD38, CD70) CD4 and proliferating (Ki67) CD4 and CD8 T cells, irrespective of previous episodes of sexually transmittable infections. Furthermore, whole blood cultures from HRSN showed lower lymphoproliferative responses than healthy laboratory controls. These data suggest that low levels of immune activation and low T cell responsiveness may contribute to low HIV susceptibility.
CD8brightCD56+ T Cells Are Cytotoxic Effectors in Patients with Active Behçet’s Uveitis
Authors: Ahn Jae Kyoun , Chung Hum , Lee Dong-sup , Yu Young Suk , Yu Hyeong Gon
Published: April 2014

Behçet’s uveitis, characterized by chronic recurrent uveitis and obliterating retinal vasculitis, frequently causes bilateral blindness. Intraocular infiltration of TCRαβ+CD8brightCD56+ cells was a distinct feature in Behçet’s uveitis. However, phenotypic natures and effector functions of the cells have remained elusive. This study was conducted to determine phenotypic and functional characteristics and cytotoxic mechanisms of CD8brightCD56+ T cells in Behçet’s uveitis. CD11b+CD27−CD62L− phenotypes of CD8brightCD56+ T cells were increased in patients with active Behçet’s uveitis compared with inactive Behcet’s patients and normal controls. Interestingly, CD45RAdimCD45RO− phenotypes were expanded, and CD94 expression was markedly up-regulated in contrast to the down-regulation of NKG2D. Furthermore, these subsets were polarized to produce IFN-γ and contained high amounts of preformed intracellular perforin while exclusively expressing surface FasL upon PI stimulation. Moreover, the cytolytic functions of freshly isolated CD8brightCD56+ T cells were up-regulated against both K562 (NK-sensitive) and Raji (NK-resistant) cells, which were effectively inhibited by perforin inhibitor (concanamycin A). Their cytolytic activity against HUVECs was also increased and was effectively suppressed by Fas ligand inhibitor (brefeldin A) and partly by perforin inhibitor. Furthermore, cytolytic functions of PMA and ionomycin-stimulated CD8brightCD56+ T cells against HUVECs were greatly enhanced, by pretreatment of recombinant human IFN-γ on HUVECs. Therefore, CD8brightCD56+ T cells in Behçet’s uveitis are characterized by cytotoxic effector phenotypes with functional NK receptors and function as strong cytotoxic effectors through both Fas ligand-dependent and perforin-dependent pathways.
Selective Activation of Peripheral Blood T Cell Subsets by Endotoxin Infusion in Healthy Human Subjects Corresponds to Differential Chemokine Activation
Authors: De Asit K. , Miller-Graziano Carol L. , Calvano Steve E. , Laudanski Krzysztof , Lowry Stephen F. , Moldawer Lyle L. , Remick Daniel G. , Rajicic Natasa , Schoenfeld David , Tompkins Ronald G.
Published: April 2014

Although activation of human innate immunity after endotoxin administration is well established, in vivo endotoxin effects on human T cell responses are not well understood. Most naive human T cells do not express receptors for LPS, but can respond to endotoxin-induced mediators such as chemokines. In this study, we characterized the in vivo response of peripheral human T cell subsets to endotoxin infusion by assessing alterations in isolated T cells expressing different phenotypes, intracellular cytokines, and systemic chemokines concentration, which may influence these indirect T cell responses. Endotoxin administration to healthy subjects produced T cell activation as confirmed by a 20% increase in intracellular IL-2, as well as increased CD28 and IL-2R α-chain (CD25) expression. Endotoxin induced indirect activation of T cells was highly selective among the T cell subpopulations. Increased IL-2 production (36.0 ± 3.7 to 53.2 ± 4.1) vs decreased IFN-γ production (33.8 ± 4.2 to 19.1 ± 3.2) indicated selective Th1 activation. Th2 produced IL-13 was minimally increased. Differentially altered chemokine receptor expression also indicated selective T cell subset activation and migration. CXCR3+ and CCR5+ expressing Th1 cells were decreased (CXCR3 44.6 ± 3.2 to 33.3 ± 4.6 and CCR5 24.8 ± 2.3 to 12 ± 1.4), whereas plasma levels of their chemokine ligands IFN-γ-inducible protein 10 and MIP-1α were increased (61.4 ± 13.9 to 1103.7 ± 274.5 and 22.8 ± 6.2 to 55.7 ± 9.5, respectively). In contrast, CCR4+ and CCR3 (Th2) proportions increased or remained unchanged whereas their ligands, eotaxin and the thymus and activation-regulated chemokine TARC, were unchanged. The data indicate selective activation among Th1 subpopulations, as well as differential Th1/Th2 activation, which is consistent with a selective induction of Th1 and Th2 chemokine ligands.
IL-17 Enhances the Net Angiogenic Activity and In Vivo Growth of Human Non-Small Cell Lung Cancer in SCID Mice through Promoting CXCR-2-Dependent Angiogenesis
Authors: Numasaki Muneo , Watanabe Mika , Suzuki Takashi , Takahashi Hidenori , Nakamura Akira , McAllister Florencia , Hishinuma Takanori , Goto Junichi , Lotze Michael T. , Kolls Jay K. , Sasaki Hidetada
Published: April 2014

In this study, we examined the biological action of IL-17 on human non-small cell lung cancer (NSCLC). Although IL-17 had no direct effect on the in vitro growth rate of NSCLC, IL-17 selectively augmented the secretion of an array of angiogenic CXC chemokines, including CXCL1, CXCL5, CXCL6, and CXCL8 but not angiostatic chemokines, by three different NSCLC lines. Endothelial cell chemotactic activity (as a measure of net angiogenic potential) was increased in response to conditioned medium from NSCLC stimulated with IL-17 compared with those from unstimulated NSCLC. Enhanced chemotactic activity was suppressed by neutralizing mAb(s) to CXCL1, CXCL5, and CXCL8 or to CXCR-2 but not to vascular endothelial growth factor-A. Transfection with IL-17 into NSCLC had no effect on the in vitro growth, whereas IL-17 transfectants grew more rapidly compared with controls when transplanted in SCID mice. This IL-17-elicited enhancement of NSCLC growth was associated with increased tumor vascularity. Moreover, treatment with anti-mouse CXCR-2-neutralizing Ab significantly attenuated the growth of both neomycin phosphotransferase gene-transfected and IL-17-transfected NSCLC tumors in SCID mice. A potential role for IL-17 in modulation of the human NSCLC phenotype was supported by the findings that, in primary NSCLC tissues, IL-17 expression was frequently detected in accumulating and infiltrating inflammatory cells and that high levels of IL-17 expression were associated with increased tumor vascularity. These results demonstrate that IL-17 increases the net angiogenic activity and in vivo growth of NSCLC via promoting CXCR-2-dependent angiogenesis and suggest that targeting CXCR-2 signaling may be a novel promising strategy to treat patients with NSCLC.
Cdc2/Cyclin B1 Interacts with and Modulates Inositol 1,4,5-Trisphosphate Receptor (Type 1) Functions
Authors: Li Xiaogui , Malathi Krishnamurthy , Krizanova Olga , Ondrias Karol , Sperber Kirk , Ablamunits Vitaly , Jayaraman Thottala
Published: April 2014

The resistance of inositol 1,4,5-trisphosphate receptor (IP3R)-deficient cells to multiple forms of apoptosis demonstrates the importance of IP3-gated calcium (Ca2+) release to cellular apoptosis. However, the specific upstream biochemical events leading to IP3-gated Ca2+ release during apoptosis induction are not known. We have shown previously that the cyclin-dependent kinase 1/cyclin B (cdk1/CyB or cdc2/CyB) complex phosphorylates IP3R1 in vitro and in vivo at Ser421 and Thr799. In this study, we show that: 1) the cdc2/CyB complex directly interacts with IP3R1 through Arg391, Arg441, and Arg871; 2) IP3R1 phosphorylation at Thr799 by the cdc2/CyB complex increases IP3 binding; and 3) cdc2/CyB phosphorylation increases IP3-gated Ca2+ release. Taken together, these results demonstrate that cdc2/CyB phosphorylation positively regulates IP3-gated Ca2+ signaling. In addition, identification of a CyB docking site(s) on IP3R1 demonstrates, for the first time, a direct interaction between a cell cycle component and an intracellular calcium release channel. Blocking this phosphorylation event with a specific peptide inhibitor(s) may constitute a new therapy for the treatment of several human immune disorders.
Cutting Edge: Apoptosis-Regulating Signal Kinase 1 Is Required for Reactive Oxygen Species-Mediated Activation of IFN Regulatory Factor 3 by Lipopolysaccharide
Authors: Chiang Edward , Dang Oanh , Anderson Keith , Matsuzawa Atsushi , Ichijo Hidenori , David Michael
Published: April 2014

IFN regulatory factor (IRF) 3 participates in the transcriptional induction of IFN-α, IFN-β, and a subset of IFN-stimulated genes (ISGs) as a result of viral infection. In addition, bacterial cell wall components such as LPS activate IRF3 in a p38-dependent manner. In this study we show that IRF3-mediated ISG induction by LPS requires the production of reactive oxygen species (ROS) by the NADPH-dependent oxidase NOX4. Furthermore, we present evidence that LPS-mediated ROS production leads to activation of apoptosis-regulating-signal kinase (ASK) 1, a MAPK kinase kinase family member capable of activating the MAP kinase 6/p38 axis. ASK1 kinase activity proved essential for IRF3-mediated ISG induction by LPS. Thus, our results presented here suggest a novel role for ROS and ASK1 in the innate immune response as signaling intermediates in the IRF3 activation pathway.
Cutting Edge: CD1a+ Antigen-Presenting Cells in Human Dermis Respond Rapidly to CCR7 Ligands
Authors: Angel Catherine E. , George Elizabeth , Brooks Anna E. S. , Ostrovsky Lena L. , Brown Tim La H. , Dunbar P. Rod
Published: April 2014

Recent data from murine models have confirmed that Langerhans cells are not the only population of APCs in the skin involved in initiating immune responses. In healthy human skin, we identify CD1a+ dermal APCs located close to the lymphatic vessels in the upper layers of the dermis that are unequivocally distinct from migrating Langerhans cells but exhibit both potent allostimulatory capacity and a chemotactic response to CCR7 ligands. In contrast, CD14+ dermal APCs are distributed throughout the dermis and lack a chemotactic response to CCR7 ligands. CD1a+ dermal APCs therefore represent an APC population distinct from Langerhans cells that are capable of migrating to lymph nodes and stimulating naive T cells. In humans, CD1a+ dermal APCs may fulfill some of the roles previously ascribed to Langerhans cells.