bluuebunny/crossref_metadata_2025 huggingface dataset:
Acquired Immune Responses to Plasmodium falciparum Merozoite Surface Protein-1 in the Human Fetus
Authors: King Christopher L. , Malhotra Indu , Wamachi Alex , Kioko John , Mungai Peter , Wahab Sherif Abdel , Koech Davy , Zimmerman Peter , Ouma John , Kazura James W.
Published: April 2014

Infants born in areas of stable malaria transmission are relatively protected against severe morbidity and high density Plasmodium falciparum blood-stage infection. This protection may involve prenatal sensitization and immunologic reactivity to malaria surface ligands that participate in invasion of red cells. We examined cord blood T and B cell immunity to P. falciparum merozoite surface protein-1 (MSP-1) in infants born in an area of stable malaria transmission in Kenya. T cell cytokine responses to the C-terminal 19-kDa fragment of MSP-1 (MSP-119) were detected in 24 of 92 (26%) newborns (4–192 IFN-γ and 3–88 IL-4-secreting cells per 106/cord blood lymphocytes). Peptide epitopes in the N-terminal block 3 region of MSP-1 also drove IFN-γ and/or IL-13 production. There was no evidence of prenatal T cell sensitization to liver-stage Ag-1. A total of 5 of 86 (6%) newborns had cord blood anti-MSP-119 IgM Abs, an Ig isotype that does not cross the placenta and is therefore of fetal origin. The frequency of neonatal B cell sensitization was higher than that indicated by serology alone, as 5 of 27 (18%) cord blood samples contained B cells that produced IgG when stimulated with MSP-119 in vitro. Neonatal B cell IgG responses were restricted to the Q-KNG allele of MSP-119, the major variant in this endemic area, whereas T cells responded to all four MSP-119 alleles evaluated. In utero sensitization to MSP-1 correlated with the presence of malaria parasites in cord blood (χ2 = 20, p < 0.0001). These data indicate that prenatal sensitization to blood-stage Ags occurs in infants born in malaria endemic areas.
IL-18 Improves the Early Antimicrobial Host Response to Pneumococcal Pneumonia
Authors: Lauw Fanny N. , Branger Judith , Florquin Sandrine , Speelman Peter , van Deventer Sander J. H. , Akira Shizuo , van der Poll Tom
Published: April 2014

To determine the role of endogenous IL-18 during pneumonia, IL-18 gene-deficient (IL-18−/−) mice and wild-type (WT) mice were intranasally inoculated with Streptococcus pneumoniae, the most common causative agent of community-acquired pneumonia. Infection with S. pneumoniae increased the expression of IL-18 mRNA and was associated with elevated concentrations of both precursor and mature IL-18 protein within the lungs. IL-18−/− mice had significantly more bacteria in their lungs and were more susceptible for progressing to systemic infection at 24 and 48 h postinoculation. Similarly, treatment of WT mice with anti-IL-18 was associated with enhanced outgrowth of pneumococci. In contrast, the clearance of pneumococci from lungs of IL-12−/− mice was unaltered when compared with WT mice. Furthermore, anti-IL-12 did not influence bacterial clearance in either IL-18−/− or WT mice. These data suggest that endogenous IL-18, but not IL-12, plays an important role in the early antibacterial host response during pneumococcal pneumonia.
Generation of Genome-Wide CD8 T Cell Responses in HLA-A*0201 Transgenic Mice by an HIV-1 Ubiquitin Expression Library Immunization Vaccine
Authors: Singh Rana A. K. , Wu Lei , Barry Michael A.
Published: April 2014

HIV-1 is a fundamentally difficult target for vaccines due to its high mutation rate and its repertoire of immunoevasive strategies. To address these difficulties, a multivalent, proteasome-targeted, live genetic vaccine was recently developed against HIV-1 using the expression library immunization approach. In this HIV-1 vaccine all open reading frames of HIV-1 are expressed from 32 plasmids as Ag fragments fused to the ubiquitin protein to increase Ag targeting to the proteasome to enhance CTL responses. In this work we demonstrate the ability of the HIV-1 library vaccine to simultaneously provoke robust HLA-A*0201-restricted T cell responses against all 32 HIV-1 library vaccine Ags after single immunization by gene gun. These CD8 T cell responses included HLA-A*0201-restricted CTL activity, CD8/IFN-γ T cell responses, and HLA tetramer binding against defined immunodominant epitopes in gag, pol, env, and nef as well as potent CD8/IFN-γ responses against undefined HLA-A*0201-restricted epitopes in all remaining Ags of the library. CD8 responses mediated by single gag, pol, env, and nef plasmids from the vaccine demonstrated little reduction in specific T cell responses when these plasmids were diluted into the context of the full 32-plasmid library, suggesting that Ag dominance or immune interference is not an overt problem to limit the efficacy of this complex vaccine. Therefore, this work demonstrates the ability of the HIV-1 library vaccine to generate robust multivalent genome-wide T cell responses as one approach to control the highly mutable and immunoevasive HIV-1 virus.
Treatment of T Cell-Dependent Experimental Colitis in SCID Mice by Local Administration of an Adenovirus Expressing IL-18 Antisense mRNA
Authors: Wirtz Stefan , Becker Christoph , Blumberg Richard , Galle Peter R. , Neurath Markus F.
Published: April 2014

Recent studies have shown that IL-18, a pleiotropic cytokine that augments IFN-γ production, is produced by intestinal epithelial cells and lamina propria cells from patients with Crohn’s disease. In this study, we show that IL-18 is strongly expressed by intestinal epithelial cells in a murine model of Crohn’s disease induced by transfer of CD62L+CD4+ T cells into SCID mice. To specifically down-regulate IL-18 expression in this model, we constructed an E1/E3-deleted adenovirus expressing IL-18 antisense mRNA, denoted Ad-asIL-18, and demonstrated the capacity of such a vector to down-regulate IL-18 expression in colon-derived DLD-1 cells and RAW264.7 macrophages. Local administration of the Ad-asIL-18 vector to SCID mice with established colitis led to transduction of epithelial cells and caused a significant suppression of colitis activity, as assessed by a newly developed endoscopic analysis system for colitis. Furthermore, treatment with Ad-asIL-18 induced a significant suppression of histologic colitis activity and caused suppression of mucosal IFN-γ production, whereas IFN-γ production by spleen T cells was unaffected. Taken together, these data indicate an important role for IL-18 in the effector phase of a T cell-dependent murine model of colitis and suggest that strategies targeting IL-18 expression may be used for the treatment of patients with Crohn’s disease.
ICA69null Nonobese Diabetic Mice Develop Diabetes, but Resist Disease Acceleration by Cyclophosphamide
Authors: Winer Shawn , Astsaturov Igor , Gaedigk Roger , Hammond-McKibben Denise , Pilon Marc , Song Aihua , Kubiak Violetta , Karges Wolfram , Arpaia Enrico , McKerlie Colin , Zucker Peter , Singh Bhagirath , Dosch H.-Michael
Published: April 2014

ICA69 (islet cell Ag 69 kDa) is a diabetes-associated autoantigen with high expression levels in β cells and brain. Its function is unknown, but knockout of its Caenorhabditis elegans homologue, ric-19, compromised neurotransmission. We disrupted the murine gene, ica-1, in 129-strain mice. These animals aged normally, but speed-congenic ICA69null nonobese diabetic (NOD) mice developed mid-life lethality, reminiscent of NOD-specific, late lethal seizures in glutamic acid decarboxylase 65-deficient mice. In contrast to wild-type and heterozygous animals, ICA69null NOD congenics fail to generate, even after immunization, cross-reactive T cells that recognize the dominant Tep69 epitope in ICA69, and its environmental mimicry Ag, the ABBOS epitope in BSA. This antigenic mimicry is thus driven by the endogenous self Ag, and not initiated by the environmental mimic. Insulitis, spontaneous, and adoptively transferred diabetes develop normally in ICA69null NOD congenics. Like glutamic acid decarboxylase 65, ICA69 is not an obligate autoantigen in diabetes. Unexpectedly, ICA69null NOD mice were resistant to cyclophosphamide (CY)-accelerated diabetes. Transplantation experiments with hemopoietic and islet tissue linked CY resistance to ICA69 deficiency in islets. CY-accelerated diabetes involves not only ablation of lymphoid cells, but ICA69-dependent drug toxicity in β cells that boosts autoreactivity in the regenerating lymphoid system.
Association of Prolonged Survival in HLA-A2+ Progressive Multifocal Leukoencephalopathy Patients with a CTL Response Specific for a Commonly Recognized JC Virus Epitope
Authors: Koralnik Igor J. , Du Pasquier Renaud A. , Kuroda Marcelo J. , Schmitz Jörn E. , Dang Xin , Zheng Yue , Lifton Michelle , Letvin Norman L.
Published: April 2014

The role of JC virus (JCV)-specific CTL was explored in the immunopathogenesis of progressive multifocal leukoencephalopathy (PML). We identified a 9-aa epitope of the JCV capsid protein VP1, the VP1p100 peptide ILMWEAVTL, which is recognized by CTL of HLA-A2+ HIV+/PML survivors. We then constructed an HLA-A*0201/VP1p100 tetrameric complex that allowed us to assess by flow cytometry the PBMC of 13 PML patients and 11 control subjects for the presence of JCV-specific CTL. VP1p100-specific CTL were detected by tetramer binding in VP1p100-stimulated PBMC of five of seven (71%) PML survivors and zero of six PML progressors (p = 0.02). Two of three HIV+ patients with a leukoencephalopathy resembling PML, but with no virologic evidence of JCV infection, also had detectable VP1p100-specific CTL in their PBMC. PBMC of eight HIV+ patients with other neurologic diseases and healthy control subjects had no detectable JCV-specific CTL. These data suggest that the JCV-specific cellular immune response may be important in the containment of PML, and the tetramer-staining assay may provide a useful prognostic tool in the clinical management of these patients.
Up-Regulation of CCR5 and CCR6 on Distinct Subpopulations of Antigen-Activated CD4+ T Lymphocytes
Authors: Ebert Lisa M. , McColl Shaun R.
Published: April 2014

Following infection, naive T cells are activated in the secondary lymphoid tissue, but then need to move to the infected tissue in the periphery to mediate their effector functions. The acquisition of inflammatory chemokine receptors, such as CCR5 and CCR6, may contribute to the efficient relocation of activated T cells to inflamed sites in the periphery. In keeping with this idea, the present study has demonstrated that CCR5 and CCR6 are up-regulated on CD4+ T cells upon activation in the MLR. The observed increase in expression correlated well with the acquisition of an activated/memory phenotype and was largely (CCR5) or completely (CCR6) separated temporally from the initiation of cell division. In contrast, the regulation of two other chemokine receptors, CXCR3 and CXCR4, occurred in close parallel with the cell division process. Increased mRNA levels are likely to contribute to the enhanced surface expression of CCR5 and CCR6, but in the case of CCR6, translocation of intracellular stores of protein to the cell surface may be an additional mechanism of regulation. The up-regulation of CCR5 was more extensive than that of CCR6, as only approximately half the activated CCR5+ T cells coexpressed CCR6. The increased expression of CCR5 resulted in enhanced chemotaxis toward the CCR5 ligand macrophage-inflammatory protein-1β/CCL4, but up-regulation of CCR6 did not result in altered chemotactic responsiveness to macrophage-inflammatory protein-3α/CCL20, suggesting an alternative function for this receptor.
Gene Expression Profiling of Mucosal Addressin Cell Adhesion Molecule-1+ High Endothelial Venule Cells (HEV) and Identification of a Leucine-Rich HEV Glycoprotein as a HEV Marker
Authors: Saito Koichi , Tanaka Toshiyuki , Kanda Hidenobu , Ebisuno Yukihiko , Izawa Dai , Kawamoto Shoko , Okubo Kosaku , Miyasaka Masayuki
Published: April 2014

High endothelial venule (HEV) cells support lymphocyte migration from the peripheral blood into secondary lymphoid tissues. Using gene expression profiling of mucosal addressin cell adhesion molecule-1+ mesenteric lymph node HEV cells by quantitative 3′-cDNA collection, we have identified a leucine-rich protein, named leucine-rich HEV glycoprotein (LRHG) that is selectively expressed in these cells. Northern blot analysis revealed that LRHG mRNA is ∼1.3 kb and is expressed in lymph nodes, liver, and heart. In situ hybridization analysis demonstrated that the mRNA expression in lymph nodes is strictly restricted to the HEV cells, and immunofluorescence analysis with polyclonal Abs against LRHG indicated that the LRHG protein is localized mainly to HEV cells and possibly to some lymphoid cells surrounding the HEVs. LRHG cDNA encodes a 342-aa protein containing 8 tandem leucine-rich repeats of 24 aa each and has high homology to human leucine-rich α2-glycoprotein. Similar to some other leucine-rich repeat protein family members, LRHG can bind extracellular matrix proteins that are expressed on the basal lamina of HEVs, such as fibronectin, collagen IV, and laminin. In addition, LRHG binds TGF-β. These results suggest that LRHG is likely to be multifunctional in that it may capture TGF-β and/or other related humoral factors to modulate cell adhesion locally and may also be involved in the adhesion of HEV cells to the surrounding basal lamina.
Heparin Induces Differentiation of CD1a+ Dendritic Cells from Monocytes: Phenotypic and Functional Characterization
Authors: Xia Chang-Qing , Kao Kuo-Jang
Published: April 2014

Dendritic cells (DCs) play important roles in initiation and regulation of immune responses. DCs derived from human monocytes can be classified according to presence of CD1a molecules. Although CD1a+ DCs can be prepared from monocytes in media containing GM-CSF, IL-4, and FCS, it has been reported that CD1a+ DCs could not be easily obtained from monocytes using media containing human serum or plasma. In this study, we demonstrate for the first time that heparin can reliably induce differentiation of CD1a+ DCs from monocytes with or without autologous serum or plasma. The development of CD1a+ DCs is heparin concentration dependent (0–50 U/ml). Comparing with CD1a− DCs developed without heparin, CD1a+ DCs express higher CD40 and CD80 and lower CD86. Both CD1a+ and CD1a− DCs express similar levels of HLA-DR. CD80, CD86, HLA-DR, and CD40 are proportionally up-regulated when both types of DCs are stimulated with LPS or LPS plus IFN-γ. The effect of heparin is neutralized by heparin-binding proteins, such as protamine sulfate, platelet factor-4, and β-thromboglobulin. Functionally, heparin-treated DCs respond to LPS or LPS plus IFN-γ with higher IL-10 and less IL-12 production than heparin-untreated DCs. Heparin-treated DCs are more potent in priming allogeneic and autologous CD4+ T cells to proliferate and to produce both type 1 and type 2 cytokines. The results of our study show that CD1a+ DCs can be prepared from monocytes ex vivo without using xenogeneic serum and may be used for immunotherapy.
Negative Regulation of CD95 Ligand Gene Expression by Vitamin D3 in T Lymphocytes
Authors: Cippitelli Marco , Fionda Cinzia , Di Bona Danilo , Di Rosa Francesca , Lupo Aldo , Piccoli Mario , Frati Luigi , Santoni Angela
Published: April 2014

Fas (APO-1/CD95) and its ligand (FasL/CD95L) are cell surface proteins whose interaction activates apoptosis of Fas-expressing targets. In T lymphocytes, the Fas/FasL system regulates activation-induced cell death, a fundamental mechanism for negative selection of immature T cells in the thymus and for maintenance of peripheral tolerance. Aberrant expression of Fas and FasL has also been implicated in diseases in which the lymphocyte homeostasis is compromised, and several studies have described the pathogenic functions of Fas and FasL in vivo, particularly in the induction/regulation of organ-specific autoimmune diseases. The 1,25(OH)2D3 is a secosteroid hormone that activates the nuclear receptor vitamin D3 receptor (VDR), whose immunosuppressive activities have been well studied in different models of autoimmune disease and in experimental organ transplantation. We and others have recently described the molecular mechanisms responsible for the negative regulation of the IFN-γ and IL-12 genes by 1,25(OH)2D3 in activated T lymphocytes and macrophages/dendritic cells. In this study, we describe the effect of 1,25(OH)2D3 on the activation of the fasL gene in T lymphocytes. We show that 1,25(OH)2D3 inhibits activation-induced cell death, fasL mRNA expression, and that 1,25(OH)2D3-activated VDR represses fasL promoter activity by a mechanism dependent on the presence of a functional VDR DNA-binding domain and ligand-dependent transcriptional activation domain (AF-2). Moreover, we identified a minimal region of the promoter containing the transcription start site and a noncanonical c-Myc-binding element, which mediates this repression. These results place FasL as a novel target for the immunoregulatory activities of 1,25(OH)2D3, and confirm the interest for a possible pharmacological use of this molecule and its derivatives.
Yersinia enterocoliticaEvasion of the Host Innate Immune Response by V Antigen-Induced IL-10 Production of Macrophages Is Abrogated in IL-10-Deficient Mice
Authors: Sing Andreas , Roggenkamp Andreas , Geiger Anna M. , Heesemann Jürgen
Published: April 2014

The virulence-associated V Ag (LcrV) of pathogenic Yersinia species is part of the translocation apparatus, required to deliver antihost effector proteins (Yersinia outer proteins) into host cells. An orthologous protein (denoted as PcrV) has also been identified in the ExoS regulon of Pseudomonas aeruginosa. Additionally, it is known that LcrV is released by yersiniae into the environment and that LcrV causes an immunosuppressive effect when injected into mice. In this study, we demonstrate for the first time that rLcrV, but not PcrV, is capable of suppressing TNF-α production in zymosan A-stimulated mouse macrophages and the human monocytic Mono-Mac-6 cell line. The underlying mechanism of TNF-α suppression could be assigned to LcrV-mediated IL (IL)-10 production, because 1) LcrV induces IL-10 release in macrophages, 2) anti-IL-10 Ab treatment completely abrogated TNF-α suppression, and 3) TNF-α suppression was absent in LcrV-treated macrophages of IL-10-deficient (IL-10−/−) mice. The relevance of LcrV-mediated immunosuppression for the pathogenicity of yersiniae became evident by experimental infection of mice; in contrast to wild-type mice, IL-10−/− mice were highly resistant against Yersinia infection, as shown by lower bacterial load in spleen and liver, absent abscess formation in these organs, and survival.
Mice Lacking Bioactive IL-12 Can Generate Protective, Antigen-Specific Cellular Responses to Mycobacterial Infection Only if the IL-12 p40 Subunit Is Present
Authors: Cooper Andrea M. , Kipnis Andre , Turner Joanne , Magram Jeanne , Ferrante Jessica , Orme Ian M.
Published: April 2014

Recent evidence suggests that absence of the IL-12p40 subunit is more detrimental to the generation of protective responses than is the absence of the p35 subunit. To determine whether this is the case in tuberculosis, both p35 and p40 knockout mice were infected with Mycobacterium tuberculosis. Mice lacking the p40 subunit were highly susceptible to increased bacterial growth, exhibited reduced production of IFN-γ, and had increased mortality. In contrast, mice lacking the p35 subunit exhibited a moderate ability to control bacterial growth, were able to generate Ag-specific IFN-γ responses, and survived infection longer. The superior Ag-specific responses of the p35 gene-disrupted mice, when compared with the p40 gene-disrupted mice, suggest that the p40 subunit may act other than as a component of IL-12. A candidate molecule capable of driving the protective responses in the p35 gene-disrupted mice is the novel cytokine IL-23. This cytokine is composed of the IL-12 p40 subunit and a p19 subunit. In support of a role for this cytokine in protective responses to M. tuberculosis, we determined that the p19 subunit is induced in the lungs of infected mice.
CXCR2 Is Necessary for the Development and Persistence of Chronic Fungal Asthma in Mice
Authors: Schuh Jane M. , Blease Kate , Hogaboam Cory M.
Published: April 2014

The role of CXCR during allergic airway and asthmatic diseases is yet to be fully characterized. Therefore, the present study addressed the role of CXCR2 during Aspergillus fumigatus-induced asthma. Mice deficient in CXCR2 (CXCR2−/−) and wild-type counterparts (CXCR2+/+) were sensitized to A. fumigatus Ags and challenged with A. fumigatus conidia, and the resulting allergic airway disease was monitored for up to 37 days. At days 3 and 7 after conidia, CXCR2−/− mice exhibited significantly greater methacholine-induced airway hyperreactivity than did CXCR2+/+ mice. In contrast, CXCR2-deficient mice exhibited significantly less airway hyperresponsiveness than the wild-type control groups at days 14 and 37 after conidia. At all times after conidia, whole lung levels of IL-4, IL-5, and eotaxin/CC chemokine ligand 11 were significantly lower in CXCR2−/− mice than in the wild-type controls. Eosinophil and T cell, but not neutrophil, recruitment into the airways of A. fumigatus-sensitized CXCR2−/− mice was significantly impaired compared with wild-type controls at all times after the conidia challenge. Whole lung levels of IFN-γ, inflammatory protein-10/CXC ligand (CXCL) 10, and monokine induced by IFN-γ (MIG)/CXCL9 were significantly increased in CXCR2−/− mice compared with CXCR2+/+ mice at various times after conidia. Interestingly, at day 3 after conidia, neutrophil recruitment and airway hyperresponsiveness in CXCR2−/− mice was mediated by inflammatory protein-10/CXCL10 and, to a lesser degree, MIG/CXCL9. Taken together, these data suggest that CXCR2 contributes to the persistence of asthmatic disease due to A. fumigatus.
Lack of CD27−CD45RA−Vγ9Vδ2+ T Cell Effectors in Immunocompromised Hosts and During Active Pulmonary Tuberculosis
Authors: Gioia Cristiana , Agrati Chiara , Casetti Rita , Cairo Cristiana , Borsellino Giovanna , Battistini Luca , Mancino Giorgio , Goletti Delia , Colizzi Vittorio , Pucillo Leopoldo P. , Poccia Fabrizio
Published: April 2014

In humans, the circulating pool of mycobacteria-reactive Vγ9Vδ2+ T cells is expanded with age and may contribute to Mycobacterium tuberculosis immunosurveillance. We observed that two subsets of Vγ9Vδ2+ T cells could be identified on the basis of CD27 expression in immunocompetent adults, showing that functionally differentiated γδ T cells have lost CD27 expression. In contrast, the CD27−CD45RA−Vγ9Vδ2+ T cell subset of effector cells was absent in cord blood cells from healthy newborns and lacking in the peripheral blood from HIV-infected patients. Moreover, circulating Vγ9Vδ2+ T cell effectors were significantly reduced in patients with acute pulmonary tuberculosis, resulting in a reduced frequency of IFN-γ-producing cells after stimulation with nonpeptidic mycobacterial ligands. These observations indicate that monitoring and boosting γδ T cell effectors could be clinically relevant both in immunocompromised hosts and during active tuberculosis disease.
Cutting Edge: Agonistic Effect of Indomethacin on a Prostaglandin D2 Receptor, CRTH2
Authors: Hirai Hiroyuki , Tanaka Kazuya , Takano Shoichi , Ichimasa Michiko , Nakamura Masataka , Nagata Kinya
Published: April 2014

Indomethacin is a widely used nonsteroidal anti-inflammatory drug and is generally known to exhibit its multiple biological functions by inhibiting cyclooxygenases or activating peroxisome proliferator-activated receptors. In this study, we present evidence demonstrating that the novel PGD2 receptor chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is another functional target for indomethacin. Indomethacin induced Ca2+ mobilization in CRTH2-transfected K562 cells at submicromolar concentrations (approximate EC50, 50 nM) in a Gαi-dependent manner as PGD2 did. Other nonsteroidal anti-inflammatory drugs (aspirin, sulindac, diclofenac, and acemetacin) had no such effect even at micromolar concentrations. In chemotaxis assay, three CRTH2-expressing cell types, Th2 cells, eosinophils, and basophils, were all significantly attracted by indomethacin (EC50, 50–500 nM) as well as by PGD2 (EC50, 2–20 nM), and the effects of indomethacin were blocked by anti-CRTH2 mAb. These results suggest the involvement of CRTH2 in mediating some of therapeutic and/or unwanted side effects of indomethacin, independently of cyclooxygenases and peroxisome proliferator-activated receptors.
Cutting Edge: Transcriptional Activity of NFATc1 Is Enhanced by the Pim-1 Kinase
Authors: Rainio Eeva-Marja , Sandholm Jouko , Koskinen Päivi J.
Published: April 2014

Pim-1 is an oncogenic serine/threonine kinase implicated in cytokine-induced signal transduction and in development of lymphoid malignancies. However, its precise function as well as physiological substrates have remained unknown. In this study we demonstrate that Pim-1 can physically interact with the NFATc1 transcription factor and phosphorylate it in vitro on several serine residues. In contrast to previously recognized NFATc kinases, wild-type Pim-1 enhances NFATc-dependent transactivation and IL-2 production in Jurkat T cells, while kinase-deficient Pim-1 mutants inhibit them in a dominant negative fashion. Our results reveal a novel, phosphorylation-dependent regulatory mechanism targeting NFATc1 through which Pim-1 acts as a downstream effector of Ras to facilitate IL-2-dependent proliferation and/or survival of lymphoid cells.
Cutting Edge: CD4 and CD8 T Cells Are Intrinsically Different in Their Proliferative Responses
Authors: Foulds Kathryn E. , Zenewicz Lauren A. , Shedlock Devon J. , Jiang Jiu , Troy Amy E. , Shen Hao
Published: April 2014

In this study, we compared the proliferation and differentiation of Ag-specific CD4 and CD8 T cells following Listeria infection. Our results show that CD4 T cells responding to infection divide a limited number of times, with progeny exhibiting proliferative arrest in early divisions. Even with increased infectious doses, CD4 T cells display this restricted proliferative pattern and are not driven to undergo extensive clonal expansion. This is in striking contrast to CD8 T cells, which undergo extensive proliferation in response to infection. These differences are also evident when CD4 and CD8 T cells receive uniform anti-CD3 stimulation in vitro. Together, these results suggest that CD4 and CD8 T cells are programmed to undergo limited and extensive proliferation, respectively, to suit their function as regulator and effector cells.
Up-Regulation of Drug Resistance-Related Vaults During Dendritic Cell Development
Authors: Schroeijers Anouk B. , Reurs Anneke W. , Scheffer George L. , Stam Anita G. M. , de Jong Mariska C. , Rustemeyer Thomas , Wiemer Erik A. C. , de Gruijl Tanja D. , Scheper Rik J.
Published: April 2014

P-glycoprotein (Pgp) and vaults are associated with multidrug resistance in tumor cells, but their physiological functions are not yet clear. Pgp, the prototypical transmembrane transporter molecule, may also facilitate the migration of skin dendritic cells (DC). Vaults—ribonucleoprotein cell organelles, frequently overexpressed in Pgp-negative drug-resistant tumor cells—have also been associated with intracellular transport processes. Given the pivotal role of DC in dealing with exposure to potentially harmful substances, the present study was set out to examine the expression of Pgp and vaults during differentiation and maturation of DC. DC were obtained from different sources, including blood-derived monocytes, CD34+ mononuclear cells, and chronic myeloid leukemia cells. Whereas flow cytometric and immunocytochemical analyses showed slightly augmented levels of Pgp, up-regulation of vault expression during DC culturing was strong, readily confirmed by Western blotting, and independent of the source of DC. In further exploring the functional significance of vault expression, it was found that supplementing DC cultures with polyclonal or mAbs against the major vault protein led to lower viabilities of LPS- or TNF-α-matured monocytes-DC. Moreover, expression of critical differentiation, maturation, and costimulatory molecules, including CD1a and CD83, was reduced and their capacity to induce Ag-specific T cell proliferative and IFN-γ release responses was impaired. These data point to a role for vaults in both DC survival and functioning as APC.
Obligatory Requirement of Sulfation for P-Selectin Binding to Human Salivary Gland Carcinoma Acc-M Cells and Breast Carcinoma ZR-75-30 Cells
Authors: Ma Yan-Qing , Geng Jian-Guo
Published: April 2014

Stimulated endothelial cells and activated platelets express P-selectin, which reacts with P-selectin glycoprotein ligand-1 (PSGL-1) for leukocyte rolling on the stimulated endothelial cells and heterotypic aggregation of the activated platelets on leukocytes. P-selectin also binds to several cancer cells in vitro and promotes the growth and metastasis of human colon carcinoma in vivo. The P-selectin/PSGL-1 interaction requires tyrosine sulfation. However, it is unknown whether sulfation is necessary for P-selectin binding to somatic cancer cells. In this study, we show that P-selectin mediated adhesion of Acc-M cells, a cell line derived from a human adenoid cystic carcinoma of salivary gland. These cells had a moderate expression of heparan sulfate-like proteoglycans, but had no detectable expressions of PSGL-1, CD24, Lewisx, and sialyl Lewisx. Treatment with sodium chlorate (a sulfation biosynthesis inhibitor), but not 4-methylumbelliferyl-β-d-xyloside (a proteoglycan biosynthesis inhibitor) or heparinases, reduced adhesion of these cells to P-selectin. Sodium chlorate also inhibited the P-selectin precipitation of the ∼160-, ∼54-, and ∼36-kDa molecules from the cell surface of Acc-M cells. Furthermore, P-selectin could bind to human breast carcinoma ZR-75-30 cells in a sulfation-dependent manner. Our results thus indicate that sulfation is essential for adhesion of nonblood-borne, epithelial-like human cancer cells to P-selectin.
Inhibitor of Apoptosis Protein fromOrgyia pseudotsugataNuclear Polyhedrosis Virus Provides a Costimulatory Signal Required for Optimal Proliferation of Developing Thymocytes
Authors: Robles María S. , Leonardo Esther , Criado Luis Miguel , Izquierdo Manuel , Martínez-A. Carlos
Published: April 2014

The inhibitors of apoptosis proteins (IAPs) constitute a family of endogenous inhibitors that control apoptosis in the cell by inhibiting caspase processing and activity. IAPs are also implicated in cell division, cell cycle regulation, and cancer. To address the role of IAPs in thymus development and homeostasis, we generated transgenic mice expressing IAP generated from the baculovirus Orgyia pseudotsugata nuclear polyhedrosis virus (OpIAP). Developing thymocytes expressing OpIAP show increased nuclear levels of NF-κB and reduced cytoplasmic levels of its inhibitor, IκBα. In mature thymocytes, OpIAP induces optimal activation and proliferation after TCR triggering in the absence of a costimulatory signal. OpIAP expression in immature thymocytes blocks TCR-induced apoptosis. Taken together, our data illustrate the pleiotropism of OpIAP in vivo.
Class II Transactivator Is Required for Maximal Expression of HLA-DOB in B Cells
Authors: Nagarajan Uma M. , Lochamy Jonathan , Chen Xinjian , Beresford Guy W. , Nilsen Roger , Jensen Peter E. , Boss Jeremy M.
Published: April 2014

HLA-DO, encoded by the HLA-DOA and HLA-DOB genes, has been shown to function as a modulator of Ag presentation. DNA microarray comparisons between B cells wild-type and mutant for the master regulator of MHC class II transcription, class II transactivator (CIITA), identified HLA-DOA and HLA-DOB as being up-regulated by CIITA. Although HLA-DOA had been shown previously to be regulated by CIITA, HLA-DOB expression was suggested to be independent of CIITA. A series of assays including quantitative RT-PCR, promoter-reporter assays, chromatin immunoprecipitations, and intracellular staining were performed to corroborate the DNA microarray analysis. The combined data demonstrate that HLA-DOB levels are increased by CIITA, and that this difference has an impact on the overall level of HLA-DO expression. Additionally, unlike the classical MHC class II genes, HLA-DOB expression was present in the absence of CIITA, indicating that additional factors mediate HLA-DOB expression in B cells.
Nasal-Associated Lymphoid Tissue Is a Mucosal Inductive Site for Virus-Specific Humoral and Cellular Immune Responses
Authors: Zuercher Adrian W. , Coffin Susan E. , Thurnheer M. Christine , Fundova Petra , Cebra John J.
Published: April 2014

Peyer’s patches are known as mucosal inductive sites for humoral and cellular immune responses in the gastrointestinal tract. In contrast, functionally equivalent structures in the respiratory tract remain elusive. It has been suggested that nasal-associated lymphoid tissue (NALT) might serve as a mucosal inductive site in the upper respiratory tract. However, typical signs of mucosal inductive sites like development of germinal center reactions after Ag stimulation and isotype switching of naive B cells to IgA production have not been directly demonstrated. Moreover, it is not known whether CTL can be generated in NALT. To address these issues, NALT was structurally and functionally analyzed using a model of intranasal infection of C3H mice with reovirus. FACS and histological analyses revealed development of germinal centers in NALT in parallel with generation and expansion of IgA+ and IgG2a+ B cells after intranasal reovirus infection. Reovirus-specific IgA was produced in both the upper respiratory and the gastrointestinal tract, whereas production of reovirus-specific IgG2a was restricted to NALT, submandibular, and mesenteric lymph nodes. Moreover, virus-specific CTL were detected in NALT. Limiting dilution analysis showed a 5- to 6-fold higher precursor CTL frequency in NALT compared with a cervical lymph node. Together these data provide direct evidence that NALT is a mucosal inductive site for humoral and cellular immune responses in the upper respiratory tract.
Recombinant Canarypox Vaccine-Elicited CTL Specific for Dominant and Subdominant Simian Immunodeficiency Virus Epitopes in Rhesus Monkeys
Authors: Santra Sampa , Schmitz Jörn E. , Kuroda Marcelo J. , Lifton Michelle A. , Nickerson Christine E. , Lord Carol I. , Pal Ranajit , Franchini Genoveffa , Letvin Norman L.
Published: April 2014

Since virus-specific CTL play a central role in containing HIV replication, a candidate AIDS vaccine should generate virus-specific CTL responses. In this study, the ability of a recombinant canarypox virus expressing SIV Gag-Pol-Env (ALVAC/SIV gag-pol-env) was assessed for its ability to elicit both dominant and subdominant epitope-specific CTL responses in rhesus monkeys. Following a series of five immunizations, memory CTL responses specific for a dominant Gag epitope could be demonstrated in the peripheral blood of vaccinated monkeys. Memory CTL responses to a subdominant Pol epitope were undetectable in these animals. Following challenge with SIVmac251, the experimentally vaccinated animals developed high frequency CTL responses specific for the dominant Gag epitope that emerged in temporal association with the early containment of viral replication. Interestingly, the experimentally vaccinated, but not the control vaccinated animals, developed CTL responses to the subdominant Pol epitope that were detectable only after containment of early viremia. Thus, recombinant canarypox vaccination elicited low frequency, but durable memory CTL populations. The temporal association of the emergence of the dominant epitope-specific response with early viral containment following challenge suggests that this immune response played a role in the accelerated clearing of early viremia in these animals. The later emerging CTL response specific for the subdominant epitope may contribute to the control of viral replication in the setting of chronic infection.
Efficient In Vivo Presentation ofListeria monocytogenes- Derived CD4 and CD8 T Cell Epitopes in the Absence of IFN-γ
Authors: Škoberne Mojca , Geginat Gernot
Published: April 2014

IFN-γ is an essential component of the early Listeria monocytogenes-specific immune response, and is also an important regulator of Ag processing and presentation. Ag presentation is required for the induction and also the effector function of antimicrobial T cells. To evaluate the effect of IFN-γ on bacterial Ag presentation in vivo, macrophages and dendritic cells were separated from L. monocytogenes-infected tissues and analyzed with peptide-specific CD4 and CD8 T cell lines in a sensitive ELISPOT-based ex vivo Ag presentation assay. The comparison of professional APCs isolated from infected IFN-γ-deficient and wild-type mice revealed different peptide presentation patterns of L. monocytogenes-derived CD8 T cell epitopes, while the presentation pattern of CD4 T cell epitopes remained unchanged. The further in vitro analysis of the generation of CD8 T cell epitopes revealed a peptide-specific effect of IFN-γ on MHC class I-restricted Ag presentation. These results show that despite this modulation of the Ag presentation pattern of CD8 T cell epitopes, IFN-γ is not generally required for the MHC class I- and MHC class II-restricted presentation of L. monocytogenes-derived antigenic peptides by professional APCs in vivo.
Recruitment of MHC Class I Molecules by Tapasin into the Transporter Associated with Antigen Processing-Associated Complex Is Essential for Optimal Peptide Loading
Authors: Tan Pamela , Kropshofer Harald , Mandelboim Ofer , Bulbuc Nadja , Hämmerling Günter J. , Momburg Frank
Published: April 2014

The ER protein tapasin (Tpn) forms a bridge between MHC class I H chain (HC)/β2-microglobulin and the TAP peptide transporter. The function of this TAP-associated complex was unclear because it was reported that soluble Tpn that has lost TAP interaction would be fully competent in terms of peptide loading and Ag presentation. We found, however, that only wild-type human Tpn (hTpn), but not three soluble hTpn variants, a transmembrane domain point mutant of hTpn (L410→F), wild-type mouse Tpn, nor a mouse-human Tpn hybrid, fully up-regulated peptide-dependent Bw4 epitopes when expressed in Tpn-deficient .220.B*4402 cells. Consistent with suboptimal peptide loading, the t1/2 of class I molecules was considerably reduced in the presence of soluble hTpn, hTpn-L410F, and murine Tpn. Furthermore, eluted peptide spectra and the class I-mediated inhibition of NK clones showed distinct differences to the hTpn transfectant. Only wild-type hTpn efficiently recruited HC and calreticulin (Crt) into complexes with TAP and endoplasmic reticulum p57 (ERp57). The L410F mutant was defective in TAP association, but bound to class I molecules, Crt, and ERp57. Mouse Tpn associated with human TAP and ERp57 on the one hand, and with HC and Crt on the other, but failed to recruit normal amounts of HLA class I molecules into the TAP complex. We conclude that the loading with peptides conferring high stability requires the Tpn-mediated introduction of HC into the TAP complex, whereas the mere interaction with Tpn is not sufficient.
Tryptase Inhibition Blocks Airway Inflammation in a Mouse Asthma Model
Authors: Oh Se-Woong , Pae Chong I. , Lee Dong-Keun , Jones Falaah , Chiang Gertrude K. S. , Kim Hwa-Ok , Moon Sung-Hwan , Cao Bolong , Ogbu Cyprian , Jeong Kwang-Won , Kozu Geoffrey , Nakanishi Hiroshi , Kahn Michael , Chi Emil Y. , Henderson William R.
Published: April 2014

Release of human lung mast cell tryptase may be important in the pathophysiology of asthma. We examined the effect of the reversible, nonelectrophilic tryptase inhibitor MOL 6131 on airway inflammation and hyper-reactivity in a murine model of asthma. MOL 6131 is a potent selective nonpeptide inhibitor of human lung mast cell tryptase based upon a β-strand template (Ki = 45 nM) that does not inhibit trypsin (Ki = 1,061 nM), thrombin (Ki = 23, 640 nM), or other serine proteases. BALB/c mice after i.p. OVA sensitization (day 0) were challenged intratracheally with OVA on days 8, 15, 18, and 21. MOL 6131, administered days 18–21, blocked the airway inflammatory response to OVA assessed 24 h after the last OVA challenge on day 22; intranasal delivery (10 mg/kg) had a greater anti-inflammatory effect than oral delivery (10 or 25 mg/kg) of MOL 6131. MOL 6131 reduced total cells and eosinophils in bronchoalveolar lavage fluid, airway tissue eosinophilia, goblet cell hyperplasia, mucus secretion, and peribronchial edema and also inhibited the release of IL-4 and IL-13 in bronchoalveolar lavage fluid. However, tryptase inhibition did not alter airway hyper-reactivity to methacholine in vivo. These results support tryptase as a therapeutic target in asthma and indicate that selective tryptase inhibitors can reduce allergic airway inflammation.
Human IgG Monoclonal Anti-αIIbβ3-Binding Fragments Derived from Immunized Donors Using Phage Display
Authors: Jacobin Marie-Josée , Laroche-Traineau Jeanny , Little Melvyn , Keller Armin , Peter Karlheinz , Welschof Martin , Nurden Alan , Clofent-Sanchez Gisèle
Published: April 2014

Previous studies of the immune response in polytransfused Glanzmann thrombasthenia (GT) patients and in autoimmune thrombocytopenic purpura (AITP) have relied on serum analysis and have shown the frequent development of Abs directed against the αIIbβ3 integrin. However, little is known about the molecular diversity of the humoral immune response to αIIbβ3 due to the paucity of mAbs issuing from these pathologies. We have isolated human IgG anti-αIIbβ3 binding fragments using combinatorial libraries of single-chain IgG created from the B cells of a GT and an AITP patient, both with serum Abs. Ab screening was performed using activated platelets or activated αIIbβ3-expressing Chinese hamster ovary cells. Sequencing of selected phage Abs showed that a broad selection of genes from virtually all V gene families had been used, indicating the diversity of the immune response. About one-half of the VH and VL segments of our IgG anti-αIIbβ3 fragments displayed extensive hypermutations in the complementarity-determining region, supporting the idea that an Ag-driven immune response was occurring in both patients. The H chain complementarity-determining region 3 analysis of phage Abs revealed motifs other than the well-known RGD and KQAGDV integrin-binding sequences. To our knowledge, our study is the first to illustrate multiple human IgG anti-αIIbβ3 reactivities and structural variations linked to the anti-platelet human immune response. Human αIIbβ3 Abs preferentially directed against the activated form of the integrin were further characterized because platelet αIIbβ3 inhibitors are potential therapeutic reagents for treating acute coronary syndromes. Currently available αIIbβ3 antagonists do not specifically recognize the activated form of the integrin.
Anti-Sm Autoantibodies in Systemic Lupus Target Highly Basic Surface Structures of Complexed Spliceosomal Autoantigens
Authors: McClain Micah T. , Ramsland Paul A. , Kaufman Kenneth M. , James Judith A.
Published: April 2014

Autoantibodies directed against spliceosomal proteins are a common and specific feature of systemic lupus erythematosus. These autoantibodies target a collection of proteins, including Sm B, B′, D1, D2, and D3. We define the common antigenic targets of Sm D2 and D3 and examine their role in spliceosomal autoimmunity. Our results define nine major common epitopes, five on Sm D2 and four on Sm D3. These epitopes have significantly higher (more basic) isoelectric points than do nonantigenic regions. In fact, this association is of sufficient power to make isoelectric point an excellent predictor of spliceosomal antigenicity. The crystallographic structure of Sm D2 and D3 is now partially described. The anti-Sm D2 and D3 antigenic targets are located on the surface of the respective three-dimensional complexed proteins, thereby suggesting that these epitopes are accessible in the native configuration. All but one of these nine epitopes conspicuously avoid the specific regions involved in intermolecular interactions within the spliceosomal complex. One of the D3 epitopes (RGRGRGMGR) has significant sequence homology with a major antigenic region of Sm D1 (containing a carboxyl-terminal glycine-arginine repeat), and anti-D3 Abs cross-react with this epitope of Sm D1. These results demonstrate that spliceosomal targets of autoimmunity are accessible on native structure surfaces and that cross-reactive epitopes, as well as structural associations of various spliceosomal Ags, may be involved in the induction of autoimmunity in systemic lupus.
Polycyclic Aromatic Hydrocarbons Affect Functional Differentiation and Maturation of Human Monocyte-Derived Dendritic Cells
Authors: Laupeze Béatrice , Amiot Laurence , Sparfel Lydie , Le Ferrec Eric , Fauchet Renée , Fardel Olivier
Published: April 2014

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BP) are environmental carcinogens exhibiting potent immunosuppressive properties. To determine the cellular bases of this immunotoxicity, we have studied the effects of PAHs on differentiation, maturation, and function of monocyte-derived dendritic cells (DC). Exposure to BP during monocyte differentiation into DC upon the action of GM-CSF and IL-4 markedly inhibited the up-regulation of markers found in DC such as CD1a, CD80, and CD40, without altering cell viability. Besides BP, PAHs such as dimethylbenz(a)anthracene and benzanthracene also strongly altered CD1a levels. Moreover, DC generated in the presence of BP displayed decreased endocytic activity. Features of LPS-mediated maturation of DC, such as CD83 up-regulation and IL-12 secretion, were also impaired in response to BP treatment. BP-exposed DC poorly stimulated T cell proliferation in mixed leukocyte reactions compared with their untreated counterparts. In contrast to BP, the halogenated arylhydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin, which shares some features with PAHs, including interaction with the arylhydrocarbon receptor, failed to phenotypically alter differentiation of monocytes into DC, suggesting that binding to the arylhydrocarbon receptor cannot mimic PAH effects on DC. Overall, these data demonstrate that exposure to PAHs inhibits in vitro functional differentiation and maturation of blood monocyte-derived DC. Such an effect may contribute to the immunotoxicity of these environmental contaminants due to the major role that DC play as potent APC in the development of the immune response.
BCR Engagement Induces Fas Resistance in Primary B Cells in the Absence of Functional Bruton’s Tyrosine Kinase
Authors: Tumang Joseph R. , Negm Robert S. , Solt Laura A. , Schneider Thomas J. , Colarusso Thomas P. , Hastings William D. , Woodland Robert T. , Rothstein Thomas L.
Published: April 2014

B cell susceptibility to Fas-mediated apoptosis is regulated in a receptor-specific fashion. CD40 engagement produces marked sensitivity to Fas killing, whereas surface Ig (sIg) engagement blocks Fas signaling for cell death in otherwise sensitive, CD40-stimulated B cell targets, and thus, induces a state of Fas resistance. The signaling mediator, Bruton’s tyrosine kinase (Btk), is required for certain sIg-triggered responses, and Btk is reported to directly bind Fas and block Fas-mediated apoptosis. For these reasons, the role of Btk as a mediator of sIg-induced Fas resistance was examined. Dysfunction of Btk through mutation, and absence of Btk through deletion did not interfere with induction of Fas resistance by anti-Ig. This may be due, at least in part, to induction of Btk-dependent Bcl-2 family members by anti-Ig after CD40 ligand treatment. However, the susceptibility to Fas-mediated apoptosis of B cell targets stimulated by CD40 ligand alone was increased in the absence of Btk. These results indicate that Fas resistance produced by sIg triggering does not require Btk, but suggests that in certain situations Btk modulates B cell susceptibility to Fas killing.