bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Aryl Hydrocarbon Receptor Signaling on the Modulation of Th1/Th2 Balance
Authors: Negishi Takaaki , Kato Yutaka , Ooneda Osamu , Mimura Junsei , Takada Tomonari , Mochizuki Hidenori , Yamamoto Masayuki , Fujii-Kuriyama Yoshiaki , Furusako Shoji
Published: April 2014

An orally active antiallergic agent, M50367, skews the Th1/Th2 balance toward Th1 dominance by suppressing naive Th cell differentiation into Th2 cells in vitro. Administration results in the suppression of IgE synthesis and peritoneal eosinophilia in vivo. In this report, we determined that M50354 (an active metabolite of M50367) was a ligand for the aryl hydrocarbon receptor (AhR); the immunological effects of this compound on in vitro Th1/Th2 differentiation from naive Th cells and Th1/Th2 balance in vivo were manifested through binding to AhR. These effects were completely abolished in AhR-deficient mice. AhR expression in the naive Th cell was significantly up-regulated by costimulation of TCR and CD28. Suppression of naive Th cell differentiation into Th2 cells via binding of M50354 to AhR was associated with inhibition of GATA-3 expression in Th cells. In addition, forced expression of a constitutively active form of AhR or activation of AhR by the addition of representative ligands suppressed naive Th cell differentiation into Th2 cells. Based on these results, we conclude that AhR functions as a modulator of the in vivo Th1/Th2 balance through activation in naive Th cells.
Human White Blood Cells Synthesize Morphine: CYP2D6 Modulation
Authors: Zhu Wei , Cadet Patrick , Baggerman Geert , Mantione Kirk J. , Stefano George B.
Published: April 2014

Human plasma contains low, but physiologically significant, concentrations of morphine that can increase following trauma or exercise. We now demonstrate that normal, human white blood cells (WBC), specifically polymorphonuclear cells, contain and have the ability to synthesize morphine. We also show that WBC express CYP2D6, an enzyme capable of synthesizing morphine from tyramine, norlaudanosoline, and codeine. Significantly, we also show that morphine can be synthesized by another pathway via l-3,4-dihydroxyphenylalanine (l-DOPA). Finally, we show that WBC release morphine into their environment. These studies provide evidence that 1) the synthesis of morphine by various animal tissues is more widespread than previously thought and now includes human immune cells. 2) Moreover, another pathway for morphine synthesis exists, via l-DOPA, demonstrating an intersection between dopamine and morphine pathways. 3) WBC can release morphine into the environment to regulate themselves and other cells, suggesting involvement in autocrine signaling since these cells express the μ3 opiate receptor subtype.
Aberrant IgM Signaling Promotes Survival of Transitional T1 B Cells and Prevents Tolerance Induction in Lupus-Prone New Zealand Black Mice
Authors: Roy Valerie , Chang Nan-Hua , Cai Yongchun , Bonventi Gabriel , Wither Joan
Published: April 2014

New Zealand Black (NZB) mice develop a lupus-like syndrome. Although the precise immune defects leading to autoantibody production in these mice have not been characterized, they possess a number of immunologic abnormalities suggesting that B cell tolerance may be defective. In the bone marrow, immature self-reactive B cells that have failed to edit their receptors undergo apoptosis as a consequence of Ig receptor engagement. Splenic transitional T1 B cells are recent bone marrow emigrants that retain these signaling properties, ensuring that B cells recognizing self-Ags expressed only in the periphery are deleted from the naive B cell repertoire. In this study we report that this mechanism of tolerance is defective in NZB mice. We show that NZB T1 B cells are resistant to apoptosis after IgM cross-linking in vitro. Although extensive IgM cross-linking usually leads to deletion of T1 B cells, in NZB T1 B cells we found that it prevents mitochondrial membrane damage, inhibits activation of caspase-3, and promotes cell survival. Increased survival of NZB T1 B cells was associated with aberrant up-regulation of Bcl-2 after Ig receptor engagement. We also show that there is a markedly increased proportion of NZB T1 B cells that express elevated levels of Bcl-2 in vivo and provide evidence that up-regulation of Bcl-2 follows encounter with self-Ag in vivo. Thus, we propose that aberrant cell signaling in NZB T1 B cells leads to the survival of autoreactive B cells, which predisposes NZB mice to the development of autoimmunity.
Short-Interfering RNA-Mediated Lck Knockdown Results in Augmented Downstream T Cell Responses
Authors: Methi Trond , Ngai Jacob , Mahic Milada , Amarzguioui Mohammed , Vang Torkel , Tasken Kjetil
Published: April 2014

The Src family kinase Lck is essential for T cell Ag receptor-mediated signaling. In this study, we report the effects of acute elimination of Lck in Jurkat TAg and primary T cells using RNA interference mediated by short-interfering RNAs. In cells with Lck knockdown (kd), proximal TCR signaling was strongly suppressed as indicated by reduced ζ-chain phosphorylation and intracellular calcium mobilization. However, we observed sustained and elevated phosphorylation of ERK1/2 in Lck kd cells 30 min to 2 h after stimulation. Downstream effects on immune function as determined by activation of a NFAT-AP-1 reporter, and TCR/CD28-stimulated IL-2 secretion were strongly augmented in Jurkat and primary T cells, respectively. As expected, overexpression of SHP-1 in Jurkat cells inhibited TCR-induced NFAT-AP-1 activation, but this effect could be overcome by simultaneous kd of Lck. Furthermore, acute elimination of Lck also suppressed TCR-mediated activation of SHP-1, suggesting the possible role of SHP-1 in a negative feedback loop originating from Lck. This report underscores Lck as an important mediator of proximal TCR signaling, but also indicates a suppressive role on downstream immune function.
Developmental Changes in the Human Heavy Chain CDR3
Authors: Souto-Carneiro M. Margarida , Sims Gary P. , Girschik Hermann , Lee Jisoo , Lipsky Peter E.
Published: April 2014

The CDR3 of the Ig H chain (CDR3H) is significantly different in fetal and adult repertoires. To understand the mechanisms involved in the developmental changes in the CDR3H of Ig H chains, sets of nonproductive VHDJH rearrangements obtained from fetal, full-term neonates and adult single B cells were analyzed and compared with the corresponding productive repertoires. Analysis of the nonproductive repertoires was particularly informative in assessing developmental changes in the molecular mechanisms of VHDJH recombination because these rearrangements did not encode a protein and therefore their distribution was not affected by selection. Although a number of differences were noted, the major reasons that fetal B cells expressed Ig H chains with shorter CDR3H were both diminished TdT activity in the DJH junction and the preferential use of the short JH proximal D segment D7–27. The enhanced usage of D7–27 by fetal B cells appeared to relate to its position in the locus rather than its short length. The CDR3H progressively acquired a more adult phenotype during ontogeny. In fetal B cells, there was decreased recurrent DJH rearrangements before VH-DJH rearrangement and increased usage of junctional microhomologies both of which also converted to the adult pattern during ontogeny. Overall, these results indicate that the decreased length and complexity of the CDR3H of fetal B cells primarily reflect limited enzymatic modifications of the joins as well as a tendency to use proximal D and JH segments during DJH rearrangements.
Distal Enhancer Elements Transcribe Intergenic RNA in the IL-10 Family Gene Cluster
Authors: Jones Elizabeth A. , Flavell Richard A.
Published: April 2014

The IL-10 gene and homologs IL-19, IL-20, and IL-24 are expressed within a highly conserved 145-kb cytokine gene cluster. Like the Th2 IL-4 cytokine gene cluster, it is feasible that there is coordinate regulation of these cytokines by distal regulatory elements spanning the locus. We initiated a search to characterize regulatory elements within the IL-10 family locus and present data herein on a conserved 40-kb region between the IL-19 and IL-10 genes. We map the location of 17 DNase I-hypersensitive sites in different murine T cell populations and identify three enhancer elements, which function in T cells in vitro. Two of these enhancer elements, located 9 kb upstream and 6.45 kb downstream of IL-10, display cell-specific function in the Th1-Th2 cell clones AE7 and D10 and also exhibit basic promoter activity. The downstream element, IL-10CNS+6.45, binds AP-1 in the absence of NFAT and expresses intergenic RNA in a Th2-specific manner, further validating its role as a Th2-specific enhancer/promoter element. We show that the five most highly conserved noncoding sequences in the 40-kb region transcribe intergenic RNA; four of these regions possess promoter activity in vitro that could account for the expression of these transcripts. Hence, we speculate that these novel regulatory elements in the IL-10 family gene locus function via an intermediate regulatory RNA.
Human Cytomegalovirus Encodes an MHC Class I-Like Molecule (UL142) That Functions to Inhibit NK Cell Lysis
Authors: Wills Mark R. , Ashiru Omodele , Reeves Matthew B. , Okecha Georgina , Trowsdale John , Tomasec Peter , Wilkinson Gavin W. G. , Sinclair John , Sissons J. G. Patrick
Published: April 2014

Clinical and low passage strains of human CMV (HCMV) encode an additional MHC class I-related molecule UL142, in addition to the previously described UL18. The UL142 open reading frame is encoded within the ULb′ region which is missing from a number of common high passage laboratory strains. Cells expressing UL142 following transfection, and fibroblasts infected with a recombinant adenovirus-expressing UL142, were used to screen both polyclonal NK cells and NK cell clones, in a completely autologous system. Analysis of 100 NK cell clones derived from five donors, revealed 23 clones that were inhibited by fibroblasts expressing UL142 alone. Small-interfering RNA-mediated knockdown of UL142 mRNA expression in HCMV-infected cells resulted in increased sensitivity to lysis. From these data we conclude that UL142 is a novel HCMV-encoded MHC class I-related molecule which inhibits NK cell killing in a clonally dependent manner.
Heterogeneous Human NK Cell Responses to Plasmodium falciparum-Infected Erythrocytes
Authors: Korbel Daniel S. , Newman Kirsty C. , Almeida Catarina R. , Davis Daniel M. , Riley Eleanor M.
Published: April 2014

Human NK cells can respond rapidly to Plasmodium falciparum-infected RBC (iRBC) to produce IFN-γ. In this study, we have examined the heterogeneity of this response among malaria-naive blood donors. Cells from all donors become partially activated (up-regulating CD69, perforin, and granzyme) upon exposure to iRBC but cells from only a subset of donors become fully activated (additionally up-regulating CD25, IFN-γ, and surface expression of lysosomal-associated membrane protein 1 (LAMP-1)). Although both CD56dim and CD56bright NK cell populations can express IFN-γ in response to iRBC, CD25 and LAMP-1 are up-regulated only by CD56dim NK cells and CD69 is up-regulated to a greater extent in this subset; by contrast, perforin and granzyme A are preferentially up-regulated by CD56bright NK cells. NK cells expressing IFN-γ in response to iRBC always coexpress CD69 and CD25 but rarely LAMP-1, suggesting that individual NK cells respond to iRBC either by IFN-γ production or cytotoxicity. Furthermore, physical contact with iRBC can, in a proportion of donors, lead to NK cell cytoskeletal reorganization suggestive of functional interactions between the cells. These observations imply that individuals may vary in their ability to mount an innate immune response to malaria infection with obvious implications for disease resistance or susceptibility.
Coordinate Expression of CC Chemokine Ligand 5, Granulysin, and Perforin in CD8+ T Cells Provides a Host Defense Mechanism againstMycobacterium tuberculosis
Authors: Stegelmann Frank , Bastian Max , Swoboda Kay , Bhat Rauf , Kiessler Viviane , Krensky Alan M. , Roellinghoff Martin , Modlin Robert L. , Stenger Steffen
Published: April 2014

The ability of CD8+ T cells to kill intracellular pathogens depends upon their capacity to attract infected cells as well as their secretion of cytolytic and antimicrobial effector molecules. We examined the Ag-induced expression of three immune effector molecules contained within cytoplasmic granules of human CD8+ T cells: the chemokine CCL5, the cytolytic molecule perforin, and the antimicrobial protein granulysin. Macrophages infected with virulent Mycobacterium tuberculosis triggered the expression of CCL5 in CD8+ T cells only in donors with previous exposure to the tuberculosis bacteria, not in naive donors. Functionally, CCL5 efficiently attracted M. tuberculosis-infected macrophages, but failed to exert direct antibacterial activity. Infected macrophages also triggered the expression of granulysin in CD8+ T cells, and granulysin was found to be highly active against drug-susceptible and drug-resistant M. tuberculosis clinical isolates. The vast majority of CCL5-positive cells coexpressed granulysin and perforin. Taken together, this report provides evidence that a subset of CD8+ T cells coordinately expresses CCL5, perforin and granulysin, thereby providing a host mechanism to attract M. tuberculosis-infected macrophages and kill the intracellular pathogen.
Toll-IL-1 Receptor Domain-Containing Adaptor Protein Is Critical for Early Lung Immune Responses againstEscherichia coliLipopolysaccharide and ViableEscherichia coli
Authors: Jeyaseelan Samithamby , Manzer Rizwan , Young Scott K. , Yamamoto Masahiro , Akira Shizuo , Mason Robert J. , Worthen G. Scott
Published: April 2014

Pulmonary bacterial diseases are a leading cause of mortality in the U.S. Innate immune response is vital for bacterial clearance from the lung, and TLRs play a critical role in this process. Toll-IL-1R domain-containing adaptor protein (TIRAP) is a key molecule in the TLR4 and 2 signaling. Despite its potential importance, the role of TIRAP-mediated signaling in lung responses has not been examined. Our goals were to determine the role of TIRAP-dependent signaling in the induction of lung innate immune responses against Escherichia coli LPS and viable E. coli, and in lung defense against E. coli in mice. LPS-induced neutrophil sequestration; NF-κB translocation; keratinocyte cell-derived chemokine, MIP-2, TNF-α, and IL-6 expression; histopathology; and VCAM-1 and ICAM-1 expression were abolished in the lungs of TIRAP−/− mice. A cell-permeable TIRAP blocking peptide attenuated LPS-induced lung responses. Furthermore, immune responses in the lungs of TIRAP−/− mice were attenuated against E. coli compared with TIRAP+/+ mice. TIRAP−/− mice also had early mortality, higher bacterial burden in the lungs, and more bacterial dissemination following E. coli inoculation. Moreover, we used human alveolar macrophages to examine the role of TIRAP signaling in the human system. The TIRAP blocking peptide abolished LPS-induced TNF-α, IL-6, and IL-8 expression in alveolar macrophages, whereas it attenuated E. coli-induced expression of these cytokines and chemokines. Taken together, this is the first study illustrating the crucial role of TIRAP in the generation of an effective early immune response against E. coli LPS and viable E. coli, and in lung defense against a bacterial pathogen.
A Model Vaccine Exploiting Fungal Mannosylation to Increase Antigen Immunogenicity
Authors: Lam Jennifer S. , Mansour Michael K. , Specht Charles A. , Levitz Stuart M.
Published: April 2014

Ag mannosylation represents a promising strategy to augment vaccine immunogenicity by targeting Ag to mannose receptors (MRs) on dendritic cells. Because fungi naturally mannosylate proteins, we hypothesized that Ags engineered in fungi would have an enhanced capacity to stimulate T cell responses. Using the model Ag OVA, we generated proteins that differentially expressed N- and O-linked mannosylation in the yeast Pichia pastoris and compared them to their unglycosylated counterparts produced in Escherichia coli. We found that yeast-derived OVA proteins containing N-linkages, extensive O-linkages, or both were more potent than the unmannosylated Ags at inducing OVA-specific CD4+ T cell proliferation. This elevated response to fungal Ags was inhibited by mannan, suggesting involvement of MRs. However, the macrophage MR (CD206) was not essential, because macrophage MR-deficient dendritic cells were fully competent in presenting yeast-derived OVA Ags. Thus, the use of fungal glycosylation to provide N-linked and/or extensive O-linked mannosylation increased the capacity of the model Ag OVA to stimulate Ag-specific T cell responses in an MR-dependent manner. These data have implications for vaccine design by providing proof of principle that yeast-derived mannosylation can enhance immunogenicity.
The Impairment of CD8 Responses Limits the Selection of Escape Mutations in Acute Hepatitis C Virus Infection
Authors: Urbani Simona , Amadei Barbara , Cariani Elisabetta , Fisicaro Paola , Orlandini Alessandra , Missale Gabriele , Ferrari Carlo
Published: April 2014

Evasion from protective CD8 responses by mutations within immunodominant epitopes represents a potential strategy of HCV persistence. To investigate the pathogenetic relevance of this mechanism, a careful search for immunodominant CD8 epitopes was conducted in six patients with chronic evolution of HCV infection by analyzing their global CD8 response with a panel of overlapping synthetic peptides covering the overall HCV sequence and by studying the CD8 frequency by tetramer staining. Immunodominant responses were followed longitudinally from the time of acute onset in relation to the evolution of the epitopic sequences. Although intensity of CD8 responses and frequency of HCV-specific CD8 cells declined over time in all patients, mutations emerged in only three of the six acute patients studied. Variant sequences were less efficiently recognized by CD8 cells than parental epitopes and were poorly efficient in inducing a CD8 response in vitro. CD8 epitopes undergoing mutations were targeted by high avidity CD8 cells more efficient in effector function. Our data support the view that immunodominant CD8 responses are affected by inhibitory mechanisms operating early after infection and that the emergence of escape mutations represents an additional mechanism of virus evasion from those CD8 responses that are functionally preserved.
Lung NF-κB Activation and Neutrophil Recruitment Require IL-1 and TNF Receptor Signaling during Pneumococcal Pneumonia
Authors: Jones Matthew R. , Simms Benjamin T. , Lupa Michal M. , Kogan Mariya S. , Mizgerd Joseph P.
Published: April 2014

Pulmonary inflammation is an essential component of the host defense against Streptococcus pneumoniae infection of the lungs. The early response cytokines, TNF-α and IL-1, are rapidly induced upon microbial exposure. Mice deficient in all TNF- and IL-1-dependent signaling receptors were used to determine the roles of these cytokines during pneumococcal pneumonia. The deficiency of signaling receptors for TNF and IL-1 decreased bacterial clearance. Neutrophil recruitment to alveolar air spaces was impaired by receptor deficiency, as was pulmonary expression of the neutrophil chemokines KC and MIP-2. Because NF-κB mediates the expression of both chemokines, we assessed NF-κB activation in the lungs. During pneumococcal pneumonia, NF-κB proteins translocate to the nucleus and activate gene expression; these functions were largely abrogated by the deficiency of receptors for TNF-α and IL-1. Thus, the combined deficiency of TNF and IL-1 signaling reduces innate immune responses to S. pneumoniae in the lungs, probably due to essential roles for these receptors in activating NF-κB.
Diversity in the Acute CD8 T Cell Response to Vaccinia Virus in Humans
Authors: Jing Lichen , Chong Tiana M. , McClurkan Christopher L. , Huang Jay , Story Brian T. , Koelle David M.
Published: April 2014

Orthopoxviruses have complex proteomes. Infection provokes a brisk CD8 response, which is required in some systems for recovery from primary infection. Little is known concerning the Ags and epitopes recognized by CD8 T cells. We examined the fine specificity of cloned and bulk human vaccinia-specific CD8 CTL by expressing polypeptide fragments from a library of vaccinia genomic DNA. This epitope discovery method emphasizes virus-specific biological activity, as the responder cells are all reactive with whole vaccinia virus. Sixteen novel epitopes, restricted by several HLA A and B alleles, were defined to the nomamer peptide level in diverse vaccinia open reading frames. An additional seven epitope were mapped to short regions of vaccinia proteins. Targets of the CD8 response included proteins assigned to structural, enzymatic, transcription factor, and immune evasion functions, and included members of all viral kinetic classes. Most epitopes were conserved in other orthopoxviruses. Responses to at least 18 epitopes were detected within a single blood sample, revealing a surprising degree of diversity. These epitopes will be useful in natural history studies of CD8 responses to vaccinia, a nonpersisting virus with long-term memory, and in the design and evaluation of attenuated and replication-incompetent vaccinia strains being tested for variola and monkeypox prevention and for the delivery of heterologous Ags.
Cationic Polypeptides Are Required for Anti-HIV-1 Activity of Human Vaginal Fluid
Authors: Venkataraman Nitya , Cole Amy L. , Svoboda Pavel , Pohl Jan , Cole Alexander M.
Published: April 2014

Mucosal surfaces of the vagina are the portals for heterosexual transmission of HIV-1 and therefore play a fundamental role in the pathogenesis of primary infection. In the search for direct biological evidence for the role of human vaginal fluid in innate host defense, we characterized the anti-HIV-1 function of cationic polypeptides within minimally manipulated vaginal fluid. In the current study we revealed that vaginal fluid confers intrinsic anti-HIV-1 properties against both X4 and R5 strains of HIV-1 and could protect against HIV-1 infection and reduce proviral genome integration in organotypic cultures of human cervicovaginal tissue. The majority of this activity was contained in the cationic polypeptide fraction, and the depletion of cationic polypeptides using a selective cation exchange resin ablated most of the intrinsic activity against HIV-1. By adding the cationic polypeptide fraction to depleted vaginal fluid, we were able to restore activity against HIV-1. Using a proteomic approach, we identified 18 cationic polypeptides within vaginal fluid, nearly all of which are either known antimicrobials or have other purported roles in host defense. Interestingly, physiologic concentrations of 13 of the cationic polypeptides were not active alone against HIV-1, yet in concert they partially restored the anti-HIV-1 activity of cation-depleted vaginal fluid. These results suggest that synergism between cationic polypeptides is complex, and full anti-HIV-1 activity probably involves the aggregate of the cationic peptides and proteins in vaginal fluid.
Human NK Cells Inhibit Cytomegalovirus Replication through a Noncytolytic Mechanism Involving Lymphotoxin-Dependent Induction of IFN-β
Authors: Iversen Ann-Charlotte , Norris Paula S. , Ware Carl F. , Benedict Chris A.
Published: April 2014

NK cells play a key role in host defense against the β-herpesvirus CMV through perforin-dependent cytolysis. In this study, we show that human NK cells can also control human CMV (HCMV) infection by a noncytolytic mechanism involving induction of IFN-β in the virus-infected cell. Both IL-2-activated primary NK cells and an IL-2-dependent NK cell line (NK-92) exhibited potent, noncytolytic anti-HCMV activity at very low E:T cell ratios (<0.1:1). Activated NK cells expressed lymphotoxin (LT)αβ on their cell surface, and secreted LTα and TNF, all of which contributed to the NF-κB-dependent release of IFN-β from infected fibroblasts. IFN-β produced by fibroblasts and NK cell-produced IFN-γ combined to inhibit HCMV replication after immediate early gene expression. These results highlight an efficient mechanism used by NK cells to activate IFN-β expression in the infected target cell that contributes to the arrest of virion production and virus spread without cellular elimination.
Preferential Targeting of CD4-CCR5 Complexes with Bifunctional Inhibitors: A Novel Approach to Block HIV-1 Infection
Authors: Mack Matthias , Pfirstinger Jochen , Haas Jürgen , Nelson Peter J. , Kufer Peter , Riethmüller Gert , Schlöndorff Detlef
Published: April 2014

Two receptors, CD4 and one of several chemokine receptors, are required for cellular HIV-1 infection, with CCR5 being the main coreceptor for macrophage-tropic strains. We have designed bifunctional fusion proteins, consisting of RANTES/CCL5 and a single-chain Fv Ab fragment against CD4 to simultaneously block CD4 and CCR5. The fusion proteins bind to both receptors, compete with RANTES/CCL5 binding, and induce down-modulation of CCR5 ∼10 times more efficiently on CD4+ compared with CD8+ T cells. Moreover, after short incubation and subsequent washout, a significant down-modulation of CCR5 was only seen with the fusion proteins and only on CD4+ cells, but not with unmodified RANTES or on CD4− cells, indicating a preferential targeting of CCR5 on CD4+ T cells. The fusion proteins block M-tropic HIV infection more efficiently than RANTES/CCL5 and CD4 Abs alone or in combination. To our knowledge this is the first report of simultaneous blockade of an HIV-1 receptor and coreceptor with bifunctional inhibitors.
Viral Inhibition of IL-1- and Neutrophil Elastase-Induced Inflammatory Responses in Bronchial Epithelial Cells
Authors: Carroll Tomás P. , Greene Catherine M. , Taggart Clifford C. , Bowie Andrew G. , O’Neill Shane J. , McElvaney Noel G.
Published: April 2014

Previously, we elucidated the intracellular mechanisms by which neutrophil elastase (NE) up-regulates inflammatory gene expression in bronchial epithelial cells. In this study, we examine the effects of both IL-1 and NE on inflammatory gene expression in 16HBE14o− bronchial epithelial cells and investigate approaches to abrogate these inflammatory responses. IL-1 induced IL-8 protein production in time- and dose-dependent fashions, an important observation given that IL-8 is a potent neutrophil chemoattractant and a key inflammatory mediator. IL-1 and NE were shown to activate the p38 MAPK pathway in 16HBE14o− cells. Western blot analysis demonstrated IL-1R-associated kinase 1 (IRAK-1) degradation in response to stimulation with both IL-1 and NE. In addition, the expression of dominant negative IRAK-1 (IRAK-1Δ), IRAK-2Δ, or IRAK-4Δ inhibited IL-1- and NE-induced NF-κB-linked reporter gene expression. Dominant negative versions of the intracellular adaptor proteins MyD88 (MyD88Δ) and MyD88 adaptor-like (Mal P/H) abrogated NE-induced NF-κB reporter gene expression. In contrast, only MyD88Δ was found to inhibit IL-1-induced NF-κB reporter activity. We also investigated the vaccinia virus proteins, A46R and A52R, which have been shown to antagonize IL-1 signaling. Transfection with A46R or A52R cDNA inhibited IL-1- and NE-induced NF-κB and IL-8R gene expression and IL-8 protein production in primary and transformed bronchial epithelial cells. Furthermore, cytokine array studies demonstrated that IL-1 and NE can up-regulate the expression of IL-6, oncostatin M, epithelial cell-derived neutrophil activating peptide-78, growth-related oncogene family members, vascular endothelial growth factor, and GM-CSF, with induction of these proteins inhibited by the viral proteins. These findings identify vaccinia virus proteins as possible therapeutic agents for the manifestations of several inflammatory lung diseases.
Effector Function of Diabetogenic CD4 Th1 T Cell Clones: A Central Role for TNF-α
Authors: Cantor Joseph , Haskins Kathryn
Published: April 2014

Effector function of T cells in autoimmune diabetes has been widely studied with mixed populations of lymphoid T cells stimulated ex vivo, but this approach does not permit evaluation of the contribution by a single T cell clone in the inflammatory site during pathogenesis. We have investigated cytokine production both in vitro and in vivo in a panel of diabetogenic CD4 Th1 T cell clones derived from the NOD mouse. SuperArray analysis showed a common pattern of mRNA expression for inflammatory cytokines and receptors in vitro after TCR stimulation. Ex vivo intracellular cytokine staining demonstrated that two important inflammatory cytokines, IFN-γ and TNF-α, were being made by these T cells recovered from the pancreas 6 days following adoptive transfer. TNF-α produced in the pancreas by pathogenic T cell clones and recruited macrophages was not the membrane-bound form. Secreted TNF-α can lead to production of multiple inflammatory chemokines, as were observed in the pathogenic clones by intracellular cytokine staining. Our results not only define the nature of an inflammatory cytokine response critical to development of diabetes, but also suggest its role in the regulation of other events during pathogenesis induced by CD4 T cells. Similar analyses in other models demonstrated that disease induced by CD4 T cell clones closely resembles spontaneous autoimmune diabetes in which both CD4 and CD8 T cells are required. Thus, cloned T cells in effect amplify effector function of T cells which otherwise may be difficult to detect without ex vivo stimulation.
In Vivo Correction of Complement Regulatory Protein Deficiency with an Inhibitor Targeting the Red Blood Cell Membrane
Authors: Spitzer Dirk , Unsinger Jacqueline , Mao Dailing , Wu Xiaobo , Molina Hector , Atkinson John P.
Published: April 2014

Because of the complement system’s involvement in many human diseases and potential complications associated with its systemic blockade, site-specific regulation of this effector system is an attractive concept. We report on further developments of such an approach using a single-chain Ab fragment as a vehicle to deliver complement regulatory proteins to a defined cell type. In a model system in which RBCs deficient in complement receptor 1-related gene/protein y (Crry) are rapidly cleared after injection into wild-type animals by a complement-dependent mechanism, we selectively reconstituted these cells with N- and C-terminally targeted recombinant forms of Crry. Transfusion of Crry-coated knockout RBCs into C57BL/6 mice extended their in vivo half-life from <5 min to ∼2 days. Maintenance of protective levels of Crry (by a combined treatment of donor and recipient RBCs) led to nearly normal RBC survival. Uniform in vitro and in vivo coating of the RBCs and the more efficient complement inhibitory capacity of C-terminally tagged Crry were other interesting features of this experimental system. These results suggest the possibility of using the single-chain Ab fragment-mediated targeting concept of complement regulatory proteins to restrict complement inhibition to the site of its excessive activation.
Cutting Edge: Lectin-Like Transcript 1 Is a Ligand for the CD161 Receptor
Authors: Aldemir Hatice , Prod’homme Virginie , Dumaurier Marie-Jeanne , Retiere Christelle , Poupon Gwenola , Cazareth Julie , Bihl Franck , Braud Veronique M.
Published: April 2014

Human NK cells and subsets of T cells or NKT cells express the orphan C-type lectin receptor CD161 (NKR-P1A) of unknown function. In contrast to rodents that possess several NKR-P1 genes coding for either activating or inhibitory receptors, the nature of signals delivered by the single human NKR-P1A receptor is still to be clarified. In this article, we show that the lectin-like transcript 1 (LLT1) molecule is a ligand for the CD161 receptor. Engagement of CD161 on NK cells with LLT1 expressed on target cells inhibited NK cell-mediated cytotoxicity and IFN-γ secretion. Conversely, LLT1/CD161 interaction in the presence of a TCR signal enhanced IFN-γ production by T cells. These findings identify a novel ligand/receptor pair that differentially regulate NK and T cell functions.
The Scaffolding Protein CG-NAP/AKAP450 Is a Critical Integrating Component of the LFA-1-Induced Signaling Complex in Migratory T Cells
Authors: El Homasany Basma Salah El Din , Volkov Yuri , Takahashi Mikiko , Ono Yoshitaka , Keryer Guy , Delouvée Annie , Looby Eileen , Long Aideen , Kelleher Dermot
Published: April 2014

T cell migration represents a complex highly coordinated process involving participation of surface receptor/ligand interactions, cytoskeletal rearrangements, and phosphorylation-dependent signaling cascades. Members of the A-kinase anchoring protein (AKAP) family of giant scaffolding proteins can assemble and compartmentalize multiple signaling and structural molecules thereby providing a platform for their targeted positioning and efficient interactions. We characterize here the expression, intracellular distribution, and functional role of the scaffolding protein CG-NAP (centrosome and Golgi localized protein kinase N-associated protein)/AKAP450 in the process of active T cell motility induced via LFA-1 integrins. This protein is predominantly localized at the centrosome and Golgi complex. T cell locomotion triggered by LFA-1 ligation induces redistribution of CG-NAP/AKAP450 along microtubules in trailing cell extensions. Using an original in situ immunoprecipitation approach, we show that CG-NAP/AKAP450 is physically associated with LFA-1 in the multimolecular signaling complex also including tubulin and the protein kinase C β and δ isoenzymes. CG-NAP/AKAP450 recruitment to this complex was specific for the T cells migrating on LFA-1 ligands, but not on the β1 integrin ligand fibronectin. Using the GFP-tagged C-terminal CG-NAP/AKAP450 construct, we demonstrate that expression of the intact CG-NAP/AKAP450 and its recruitment to the LFA-1-associated multimolecular complex is critically important for polarization and migration of T cells induced by this integrin.
Silencing Human NKG2D, DAP10, and DAP12 Reduces Cytotoxicity of Activated CD8+ T Cells and NK Cells
Authors: Karimi Mobin , Cao Thai M. , Baker Jeanette A. , Verneris Michael R. , Soares Luis , Negrin Robert S.
Published: April 2014

Human CD8+ T cells activated and expanded by TCR cross-linking and high-dose IL-2 acquire potent cytolytic ability against tumors and are a promising approach for immunotherapy of malignant diseases. We have recently reported that in vitro killing by these activated cells, which share phenotypic and functional characteristics with NK cells, is mediated principally by NKG2D. NKG2D is a surface receptor that is expressed by all NK cells and transmits an activating signal via the DAP10 adaptor molecule. Using stable RNA interference induced by lentiviral transduction, we show that NKG2D is required for cytolysis of tumor cells, including autologous tumor cells from patients with ovarian cancer. We also demonstrated that NKG2D is required for in vivo antitumor activity. Furthermore, both activated and expanded CD8+ T cells and NK cells use DAP10. In addition, direct killing was partially dependent on the DAP12 signaling pathway. This requirement by activated and expanded CD8+ T cells for DAP12, and hence stimulus from a putative DAP12-partnered activating surface receptor, persisted when assayed by anti-NKG2D Ab-mediated redirected cytolysis. These studies demonstrated the importance of NKG2D, DAP10, and DAP12 in human effector cell function.
CD80 Cytoplasmic Domain Controls Localization of CD28, CTLA-4, and Protein Kinase Cθ in the Immunological Synapse
Authors: Tseng Su-Yi , Liu Mengling , Dustin Michael L.
Published: April 2014

The binding of costimulatory ligand CD80 to CD28 or CTLA-4 on T cells plays an important role in the regulation of the T cell response. We have examined the role of the cytoplasmic domain of CD80 in murine T cell costimulation and its organization in the immunological synapse (IS). Removal of CD80 cytoplasmic tail decreased its effectiveness in costimulating T cell proliferative response and early IL-2 production in response to agonist MHC-peptide complexes. Immunofluorescent study showed a decreased tailless CD80 accumulation in the IS of naive T cells. The two forms of CD80 accumulated differently at the IS; the tailless CD80 was colocalized with the TCR whereas the full-length CD80 was segregated from the TCR. In addition, we showed that CD80, CD28, and protein kinase Cθ colocalized in the presence or absence of the CD80 cytoplasmic tail. Thus, the cytoplasmic tail of CD80 regulates its spatial localization at the IS and that of its receptors and T cell signaling molecules such as protein kinase Cθ, and thereby facilitates full T cell activation.
Cooperation of Invariant NKT Cells and CD4+CD25+ T Regulatory Cells in the Prevention of Autoimmune Myasthenia
Authors: Liu Ruolan , La Cava Antonio , Bai Xue-Feng , Jee Youngheun , Price Mary , Campagnolo Denise I. , Christadoss Premkumar , Vollmer Timothy L. , Van Kaer Luc , Shi Fu-Dong
Published: April 2014

CD1d-restricted NKT cells and CD4+CD25+ regulatory T (Treg) cells are thymus-derived subsets of regulatory T cells that have an important role in the maintenance of self-tolerance. Whether NKT cells and Treg cells cooperate functionally in the regulation of autoimmunity is not known. We have explored this possibility in experimental autoimmune myasthenia gravis (EAMG), an animal model of human myasthenia gravis, induced by immunization of C57BL/6 mice with the autoantigen acetylcholine receptor. We have demonstrated that activation of NKT cells by a synthetic glycolipid agonist of NKT cells, α-galactosylceramide (α-GalCer), inhibits the development of EAMG. α-GalCer administration in EAMG mice increased the size of the Treg cell compartment, and augmented the expression of foxp3 and the potency of CD4+CD25+ cells to inhibit proliferation of autoreactive T cells. Furthermore, α-GalCer promoted NKT cells to transcribe the IL-2 gene and produce IL-2 protein. Depletion of CD25+ cells or neutralization of IL-2 reduced the therapeutic effect of α-GalCer in this model. Thus, α-GalCer-activated NKT cells can induce expansion of CD4+CD25+ Treg cells, which in turn mediate the therapeutic effects of α-GalCer in EAMG. Induced cooperation of NKT cells and Treg cells may serve as a superior strategy to treat autoimmune disease.
CD4+ T Cell Responses Elicited by Different Subsets of Human Skin Migratory Dendritic Cells
Authors: Morelli Adrian E. , Rubin J. Peter , Erdos Geza , Tkacheva Olga A. , Mathers Alicia R. , Zahorchak Alan F. , Thomson Angus W. , Falo Louis D. , Larregina Adriana T.
Published: April 2014

Skin dendritic cells (DC) are professional APC critical for initiation and control of adaptive immunity. In the present work we have analyzed the CD4+ T cell stimulatory function of different subsets of DC that migrate spontaneously from human skin explants, including CD1a+CD14− Langerhans’ cells (LC), CD1a−CD14− dermal DC (DDC), and CD1a−CD14+ LC precursors. Skin migratory DC consisted of APC at different stages of maturation-activation that produced IL-10, TGF-β1, IL-23p19, and IL-12p40, but did not release IL-12p70 even after exposure to DC1-driving stimuli. LC and DDC migrated as mature/activated APC able to stimulate allogeneic naive CD4+ T cells and to induce memory Th1 cells in the absence of IL-12p70. The potent CD4+ T cell stimulatory function of LC and DDC correlated with their high levels of expression of MHC class II, adhesion, and costimulatory molecules. The Th1-biasing function of LC and DDC depended on their ability to produce IL-23. By contrast, CD1a−CD14+ LC precursors migrated as immature-semimature APC and were weak stimulators of allogeneic naive CD4+ T cells. However, and opposite of a potential tolerogenic role of immature DC, the T cell allostimulatory and Th1-biasing function of CD14+ LC precursors increased significantly by augmenting their cell number, prolonging the time of interaction with responding T cells, or addition of recombinant human IL-23 in MLC. The data presented in this study provide insight into the function of the complex network of skin-resident DC that migrate out of the epidermis and dermis after cutaneous immunizations, pathogen infections, or allograft transplantation.
Stabilized β-Catenin Extends Thymocyte Survival by Up-Regulating Bcl-xL
Authors: Xie Huimin , Huang Zhaofeng , Sadim Maureen S. , Sun Zuoming
Published: April 2014

CD4+CD8+ double-positive (DP) thymocytes, which are extremely sensitive to apoptosis, specifically up-regulate Bcl-xL to extend their lifespan. Deletion of the Bcl-xL gene leads to premature apoptosis of the thymocytes. In this study, we show that stabilization of β-catenin, a critical coactivator for T cell factor (TCF), enhances DP thymocyte survival via up-regulating Bcl-xL. Spontaneous or glucocorticoid-induced thymocyte apoptosis was associated with reduced levels of β-catenin and Bcl-xL. Transgenic expression of a stabilized β-catenin protected DP thymocytes from both spontaneous and glucocorticoid-induced apoptosis, resulting in significantly increased thymic cellularity. Compared with the wild-type mice, both protein and transcript levels of Bcl-xL were significantly increased in thymocytes of β-catenin transgenic mice. In addition, TCF-1 as well as β-catenin were able to stimulate transcriptional activity of the reporter driven by a Bcl-xL promoter. β-Catenin/TCF is thus able to act as a signal to up-regulate Bcl-xL levels in DP thymocytes, resulting in their enhanced survival.
The Cytotoxicity Receptor CRACC (CS-1) Recruits EAT-2 and Activates the PI3K and Phospholipase Cγ Signaling Pathways in Human NK Cells
Authors: Tassi Ilaria , Colonna Marco
Published: April 2014

The CD2-like receptor-activating cytotoxic cell (CRACC) is a cell surface receptor of the CD2 family that triggers NK cell-mediated cytotoxicity through an undefined signaling pathway. CRACC contains cytoplasmic tyrosine-based motifs, immunoreceptor tyrosine-based switch motifs, which resemble those found in the NK cell receptor 2B4. In 2B4, these motifs recruit the adaptor signaling lymphocytic activation molecule-associated protein (SAP), which initiates a signaling cascade mediating cytotoxicity. However, CRACC does not recruit SAP. In this study, we demonstrate that, upon activation, CRACC associates with a homolog of SAP, Ewing’s sarcoma’s/FLI1-activated transcript 2 (EAT-2), in human NK cells. We show that association of EAT-2 induces the phosphorylation of CRACC and that this process is partially reduced by a pharmacological inhibitor of Src kinases. We identify PLCγ1, PLCγ2, and PI3K as the major signaling mediators downstream of CRACC/EAT-2 implicated in NK cell-mediated cytotoxicity. Moreover, EAT-2 also associates with 2B4 predominantly in resting NK cells, whereas SAP preferentially binds 2B4 upon activation. These results outline a new signaling pathway that triggers CRACC-mediated cytotoxicity and modulates 2B4-mediated activation.
CD43 Functions as a Ligand for E-Selectin on Activated T Cells
Authors: Matsumoto Masanori , Atarashi Kazuyuki , Umemoto Eiji , Furukawa Yuko , Shigeta Akiko , Miyasaka Masayuki , Hirata Takako
Published: April 2014

E-selectin, an inducible cell adhesion molecule expressed on endothelial cells, mediates the rolling on endothelium of leukocytes expressing E-selectin ligands, such as neutrophils and activated T cells. Although previous studies using mice lacking P-selectin glycoprotein ligand-1 (PSGL-1) have indicated that PSGL-1 on Th1 cells functions as an E-selectin ligand, the molecular nature of E-selectin ligands other than PSGL-1 remains unknown. In this study, we show that a 130-kDa glycoprotein was precipitated by an E-selectin-IgG chimera from mouse Th1 cells. This protein was cleaved by O-sialoglycoprotein endopeptidase and required sialic acid for E-selectin binding. The mAb 1B11, which recognizes the 130-kDa glycoform of CD43, recognized the 130-kDa band in the E-selectin-IgG precipitate. In addition, immunoprecipitation of the E-selectin-IgG precipitate with 1B11 depleted the 130-kDa protein, further confirming its identity as CD43. CD43 was also precipitated with E-selectin-IgG from cultured human T cells. E-selectin-dependent cell rolling on CD43 was observed under flow conditions using a CD43-IgG chimera generated in Chinese hamster ovary cells expressing α-1,3-fucosyltransferase VII and a core 2 β-1,6-N-acetylglucosaminyltransferase. These results suggest that CD43, when modified by a specific set of glycosyltranferases, can function as an E-selectin ligand and therefore potentially mediate activated T cell migration into inflamed sites.
Elevated T Regulatory Cells in Long-Term Stable Transplant Tolerance in Rhesus Macaques Induced by Anti-CD3 Immunotoxin and Deoxyspergualin
Authors: Asiedu Clement K. , Goodwin Karen J. , Balgansuren Gansuvd , Jenkins Stacie M. , Le Bas-Bernardet Stéphanie , Jargal Uuganbayar , Neville David M. , Thomas Judith M.
Published: April 2014

Regulatory T cells (Tregs) are implicated in immune tolerance and are variably dependent on IL-10 for in vivo function. Brief peritransplant treatment of multiple nonhuman primates (NHP) with anti-CD3 immunotoxin and deoxyspergualin has induced stable (5–10 years) rejection-free tolerance to MHC-mismatched allografts, which associated with sustained elevations in serum IL-10. In this study, we demonstrate that resting and activated PBMC from long-term tolerant NHP recipients are biased to secrete high levels of IL-10, compared with normal NHP PBMC. Although IL-10-producing CD4+ Tregs (type 1 regulatory cells (TR1)/IL-10 Tregs) were undetectable (<0.5%) in normal rhesus monkeys, 7.5 ± 1.7% of circulating CD4+ T cells of tolerant rhesus recipients expressed IL-10. In addition to this >15-fold increase in Tr1/IL-10 Tregs, the tolerant monkeys exhibited a nearly 3-fold increase in CD4+CD25+ Tregs, 8.1 ± 3.0% of CD4 T cells vs 2.8 ± 1.4% in normal cohorts (p < 0.02). The frequency of CD4+CD25+IL-10+ cells was elevated 5-fold in tolerant vs normal NHP (1.8 ± 0.9% vs 0.4 ± 0.2%). Rhesus CD4+CD25+ Tregs exhibited a memory phenotype, and expressed high levels of Foxp3 and CTLA-4 compared with CD4+CD25− T cells. Also, NHP CD4+CD25+ Tregs proliferated poorly after activation and suppressed proliferation of CD4+CD25− effector T cells, exhibiting regulatory properties similar to rodent and human CD4+CD25+ Tregs. Of note, depletion of CD4+CD25+ Tregs restored indirect pathway antidonor responses in tolerant NHP. Our study demonstrates an expanded presence of Treg populations in tolerant NHP recipients, suggesting that these adaptations may be involved in maintenance of stable tolerance.