bluuebunny/crossref_metadata_2025 huggingface dataset:
Respiratory Infection with Influenza A Virus Interferes with the Induction of Tolerance to Aeroallergens
Authors: Tsitoura Daphne C. , Kim Sung , Dabbagh Karim , Berry Gerald , Lewis David B. , Umetsu Dale T.
Published: April 2014

Viral respiratory infections have been implicated in influencing allergen sensitization and the development of asthma, but their exact role remains controversial. Because respiratory exposure to Ag normally engenders T cell tolerance and prevents the development of airway hyperreactivity (AHR) and inflammation, we examined the effects of influenza A virus infection on tolerance induced by exposure to intranasal (i.n.) OVA and the subsequent development of AHR. We found that concurrent infection with influenza A abrogated tolerance induced by exposure to i.n. OVA, and instead led to the development of AHR accompanied by the production of OVA-specific IgE, IL-4, IL-5, IL-13, and IFN-γ. When both IL-4 and IL-5 were neutralized in this system, AHR was still induced, suggesting that influenza-induced cytokines such as IL-13, or mechanisms unrelated to cytokines, might be responsible for the development of AHR. The length of time between influenza A infection and i.n. exposure to OVA was crucial, because mice exposed to i.n. OVA 15–30 days after viral inoculation developed neither AHR nor OVA-specific tolerance. These mice instead acquired Th1-biased OVA-specific immune responses associated with vigorous OVA-induced T cell proliferation, and reduced production of OVA-specific IgE. The protective effect of influenza A on AHR was dependent on IFN-γ, because protection was abrogated with a neutralizing anti-IFN-γ mAb. These results suggest that viral respiratory infection interferes with the development of respiratory allergen-induced tolerance, and that the time interval between viral infection and allergen exposure is critical in determining whether viral infection will enhance, or protect against, the development of respiratory allergen sensitization and AHR.
Host CD40 Ligand Deficiency Induces Long-Term Allograft Survival and Donor-Specific Tolerance in Mouse Cardiac Transplantation But Does Not Prevent Graft Arteriosclerosis
Authors: Shimizu Koichi , Schönbeck Uwe , Mach François , Libby Peter , Mitchell Richard N.
Published: April 2014

Although interruption of CD40-CD40L interactions via their respective mAbs yields prolonged allograft survival, the relative importance of CD40 or CD40L on donor or host cells remains unknown. Moreover, it is uncertain whether any allospecific tolerance occurring with CD40-CD40L blockade will also prevent allograft arteriopathy, the major long-term limitation to transplantation. Therefore, we performed cardiac transplantations using CD40L-deficient (CD40L−/−) mice to investigate the mechanisms underlying prolonged allograft survival. Without immunosuppression, wild-type (WT) hosts rejected allo-mismatched WT or CD40L−/− heart allografts within 2 wk. Conversely, allografts in CD40L−/− hosts beat vigorously for 12 wk. Anti-CD40 treatment did not induce graft failure in CD40L−/− recipients. Although graft-infiltrating cells were reduced ∼50% in CD40L−/− hosts, the relative percentages of macrophages and T cell subsets were comparable to WT. IFN-γ, TNF-α, and IL-10 were diminished commensurate with the reduced cellular infiltrate; IL-4 was not detected. CD40L−/− recipients did not develop IgG alloantibodies and showed diminished B7 and CD28 expression on subsets of graft-infiltrating cells. CD40L−/− transplant recipients developed allospecific tolerance to the donor haplotype; second set donor skin grafts engrafted well, whereas third-party skin grafts were vigorously rejected. By MLR, splenocytes from CD40L−/− allograft recipients also demonstrated allo-specific hyporesponsiveness. Nevertheless, allografts in CD40L−/− hosts developed significant graft arteriosclerosis by 8–12 wk posttransplant. Therefore, we propose that early alloresponses, without CD40-CD40L costimulation, induce allospecific tolerance but may trigger allo-independent mechanisms that ultimately result in graft vasculopathy.
Cutting Edge: Lipopolysaccharide Induces Physical Proximity Between CD14 and Toll-Like Receptor 4 (TLR4) Prior to Nuclear Translocation of NF-κB
Authors: Jiang Qingqi , Akashi Sachiko , Miyake Kensuke , Petty Howard R.
Published: April 2014

CD14, a GPI-linked protein, plays a pivotal role in LPS-mediated signaling by potentiating leukocyte adherence, activation, and cytokine production. Recent studies have identified the Toll-like receptor 4 (TLR4) as a membrane cofactor in LPS-mediated transmembrane signaling in cytokine induction, although the mechanism responsible for this cooperation is unknown. Using fluorescence resonance energy transfer (RET) techniques, we demonstrate that LPS triggers a physical association between CD14 and TLR4. Because LPS stimulation up-regulates CD14 and TLR4 expression, it was necessary to control for the possibility that these newly expressed molecules were associated with one another independent of LPS stimulation. Although the calcium ionophore A23187 increased the expression of CD14 and TLR4, they did not exhibit energy transfer. However, following A23187 treatment, LPS promoted physical proximity between CD14 and TLR4. Therefore, we suggest that a close interaction between CD14 and TLR4 participates in LPS signaling, leading to nuclear translocation of NF-κB.
Cutting Edge: NK Cell Inhibitory Receptors Prevent Tyrosine Phosphorylation of the Activation Receptor 2B4 (CD244)
Authors: Watzl Carsten , Stebbins Christopher C. , Long Eric O.
Published: April 2014

2B4 is an NK cell activation receptor that can provide a costimulatory signal to other activation receptors and whose mode of signal transduction is still unknown. We show that cross-linking of 2B4 on NK cells results in its rapid tyrosine phosphorylation, implying that this initial step in 2B4 signaling does not require coligation of other receptors. Ligation of 2B4 in the context of an NK cell-target cell interaction leads to 2B4 tyrosine phosphorylation, target cell lysis, and IFN-γ release. Coligation of 2B4 with the inhibitory receptors killer cell Ig-like receptor (KIR)2DL1 or CD94/NKG2 completely blocks NK cell activation. The rapid tyrosine phosphorylation of 2B4 observed upon contact of NK cells with sensitive target cells is abrogated when KIR2DL1 or CD94/NKG2 are engaged by their cognate MHC class I ligand on resistant target cells. These results demonstrate that NK inhibitory receptors can interfere with a step as proximal as phosphorylation of an activation receptor.
Differential Requirement of ZAP-70 for CD2-Mediated Activation Pathways of Mature Human T Cells
Authors: Meinl Edgar , Lengenfelder Doris , Blank Norbert , Pirzer Rainer , Barata Luis , Hivroz Claire
Published: April 2014

This study addresses the role of the tyrosine kinase ZAP-70 in CD2-mediated T cell activation. Patients lacking ZAP-70 have few mature CD8+ T cells and high numbers of CD4+ T cells that are nonfunctional upon TCR triggering. Such a patient with a homozygous deletion in the zap-70 gene that resulted in the complete absence of ZAP-70 protein expression has been identified. Expression of the tyrosine kinases Lck, Fyn, and Syk was normal. The patient’s T cells were activated with two different pairs of mitogenic mAbs. CD2-induced phosphorylation of the ζ-chain and influx of Ca2+ was defective in the ZAP-70-deficient T cells, whereas CD2-induced phosphorylation of several other proteins, including Syk, was not affected. CD2-induced proliferation as well as production of TNF-α and IFN-γ was abrogated in ZAP-70-deficient T cells, whereas PMA plus ionomycin induced normal activation of these cells. Together, this study shows that CD2-activation triggers ZAP-70-dependent and -independent pathways. Deletion of ZAP-70 affected CD2- and CD3-mediated proliferation and cytokine production in a similar way, suggesting that one of the different CD2 pathways converges with a CD3 pathway at or upstream of the activation of ZAP-70.
Prolonged Exposure to Lipopolysaccharide Inhibits Macrophage 5-Lipoxygenase Metabolism Via Induction of Nitric Oxide Synthesis
Authors: Coffey Michael J. , Phare Susan M. , Peters-Golden Marc
Published: April 2014

LPS from bacteria can result in the development of sepsis syndrome and acute lung injury. Although acute exposure to endotoxin primes leukocytes for enhanced synthesis of leukotrienes (LT), little is known about the effect of chronic exposure. Therefore, we determined the effect of prolonged LPS treatment on 5-lipoxygenase (5-LO) metabolism of arachidonic acid in alveolar macrophages (AM) and in peripheral blood monocytes. Pretreatment of AM with LPS caused time- and dose-dependent suppression of LT synthetic capacity. LPS pretreatment failed to inhibit arachidonic acid (AA) release. The fact that LPS inhibited LT synthesis from endogenous AA more than from exogenous AA suggested an effect on 5-LO-activating protein (FLAP). In addition, an inhibitory effect of LPS treatment on AM 5-LO activity was suggested by cell-free 5-LO enzyme assay. No effect on the expression of either 5-LO or FLAP proteins was observed. New protein synthesis was necessary for LPS-induced reduction of 5-LO metabolism in AM, and immunoblotting demonstrated marked induction of NO synthase (NOS). Inhibition by LPS was reproduced by an NO donor and was abrogated by inhibitors of constitutive and inducible NOS. Compared with AM, peripheral blood monocytes exhibited no suppression by LPS of 5-LO metabolism and no induction of inducible NOS. We conclude that prolonged exposure to LPS impairs AM 5-LO metabolism by NO-mediated suppression of both 5-LO and FLAP function. Because LT contribute to antimicrobial defense, this down-regulation of 5-LO metabolism may contribute to the increased susceptibility to pneumonia in patients following sepsis.
CD4 Help-Independent Induction of Cytotoxic CD8 Cells to Allogeneic P815 Tumor Cells Is Absolutely Dependent on Costimulation
Authors: Zhan Yifan , Corbett Alexandra J. , Brady Jamie L. , Sutherland Robyn M. , Lew Andrew M.
Published: April 2014

Mice made transgenic (Tg) for a rat anti-mouse CD4 Ab (GK mice) represent a novel CD4-deficient model. They not only lack canonical CD4 cells in the periphery, but also lack the residual aberrant Th cells that are found in CD4−/− mice and MHC class II−/− mice. To analyze the role of CD4 help and costimulation for CTL induction against alloantigens, we have assessed the surface and functional phenotype of CD8 cells in vivo (e.g., clearance of allogeneic P815 cells) and in vitro. In our CD4-deficient GK mice, CTL responses to allogeneic P815 cells were induced, albeit delayed, and were sufficient to eliminate P815 cells. Induction of CTL and elimination of allogeneic P815 cells were inhibited both in the presence and absence of CD4 cells by temporary CD40 ligand blockade. This indicated that direct interaction of CD40/CD40L between APCs and CD8 cells may be an accessory signal in CTL induction (as well as the indirect pathway via APC/CD4 interaction). Furthermore, whereas in CTLA4Ig single Tg mice P815 cells were rejected promptly, in the double Tg GK/CTLA4Ig mice CTL were not induced and allogeneic P815 cells were not rejected. These findings suggest that CD40/CD40L is involved in both CD4-dependent and CD4-independent pathways, and that B7/CD28 is pivotal in the CD4-independent pathway of CTL induction against allogeneic P815 cells.
Conventional, Naive CD4+ T Cells Provide an Initial Source of IL-4 During Th2 Differentiation
Authors: Noben-Trauth Nancy , Hu-Li Jane , Paul William E.
Published: April 2014

IL-4 is known to promote the differentiation of CD4+ T cells into IL-4-secreting Th2 cells. However, the cellular source of the early burst of IL-4 that drives Th2 responses in vivo has not been conclusively identified. Mice deficient for the IL-4 receptor α-chain (IL-4Rα−/−) retain the capacity to secrete IL-4 and can be used to identify those cell types that produce IL-4 without a requirement for prior IL-4-mediated stimulation. To address whether naive, conventional CD4+ T cells may act as initial producers of IL-4 in Ag-specific responses, we crossed the BALB/c IL-4Rα−/−mice to DO11.10/scid TCR transgenic mice. Lymph node cells from wild-type and IL-4Rα−/− DO11.10/scid mice secreted ∼50 pg of IL-4 per106 cells within 48 h after peptide stimulation. This small amount of IL-4 was sufficient to cause the differentiation of wild-type CD4+ T cells into Th2 cells, particularly if IFN-γ and IL-12 were neutralized during the priming cultures. CD4+ cells from the IL-4Rα−/− mice gave rise to a minor proportion (∼2%) of IL-4-producing cells upon stimulation in the presence of anti-IFN-γ and anti-IL-12. These data show that conventional, naive CD4+ T cells may be considered as initial sources of IL-4 and, in the absence of IFN-γ and IL-12, this IL-4 can induce Th2 polarization.
Differential Survival of Naive CD4 and CD8 T Cells
Authors: Ferreira Cristina , Barthlott Thomas , Garcia Sylvie , Zamoyska Rose , Stockinger Brigitta
Published: April 2014

In this paper we compare survival characteristics of transgenic and polyclonal CD4 and CD8 T cells. Transgenic CD4 T cells have an intrinsically lower capacity for survival, reflected in their gradual disappearance in thymectomized hosts, their increased sensitivity to apoptosis in vitro, and fewer divisions during homeostatic proliferation upon transfer into syngeneic lymphopenic hosts compared with CD8 T cells. Homeostatic proliferation, however, does not generally result in phenotypic conversion of activation markers unless cognate or cross-reactive Ag is present. T cells from the A18 TCR transgenic strain normally selected into the CD4 lineage are fragile as CD4 T cells, yet display the typical robust survival pattern of CD8 T cells when diverted into the CD8 lineage in a CD4-deficient host. Polyclonal CD4 and CD8 T cells also show distinctive patterns of survival, emphasizing that survival signals are relayed differently in the two lymphocyte subpopulations. However, expression levels of Bcl-2 in either transgenic or polyclonal naive CD4 and CD8 T cells are similar, excluding a role for this molecule as a key factor in differential survival of CD4 vs CD8 T cells.
Development of Dendritic Epidermal T Cells with a Skewed Diversity of γδTCRs in Vδ1-Deficient Mice
Authors: Hara Hiromitsu , Kishihara Kenji , Matsuzaki Goro , Takimoto Hiroaki , Tsukiyama Tadasuke , Tigelaar Robert E. , Nomoto Kikuo
Published: April 2014

One of the most intriguing features of γδ T cells that reside in murine epithelia is the association of a specific Vγ/Vδ usage with each epithelial tissue. Dendritic epidermal T cells (DETCs) in the murine epidermis, are predominantly derived from the “first wave” Vγ5+ fetal thymocytes and overwhelmingly express the canonical Vγ5/Vδ1-TCRs lacking junctional diversity. Targeted disruption of the Vδ1 gene resulted in a markedly impaired development of Vγ5+ fetal thymocytes as precursors of DETCs; however, γδTCR+ DETCs with a typical dendritic morphology were observed in Vδ1−/− mice and their cell densities in the epidermis were slightly lower than those in Vδ1+/− epidermis. Moreover, the Vδ1-deficient DETCs were functionally competent in their ability to up-regulate cytokines and keratinocyte growth factor-expression in response to keratinocytes. Vγ5+ DETCs were predominant in the Vδ1−/− epidermis, though Vγ5− γδTCR+ DETCs were also detected. The Vγ5+ DETCs showed a typical dendritic shape, γδTCRhigh, and age-associated expansion in epidermis as observed in conventional DETCs of normal mice, whereas the Vγ5− γδTCR+ DETCs showed a less dendritic shape, γδTCRlow, and no expansion in the epidermis, consistent with their immaturity. These results suggest that optimal DETC development does not require a particular Vγ/Vδ-chain usage but requires expression of a limited diversity of γδTCRs, which allow DETC precursors to mature and expand within the epidermal microenvironment.
Thymocyte Maturation: Selection for In-Frame TCR α-Chain Rearrangement Is Followed by Selection for Shorter TCR β-Chain Complementarity-Determining Region 3
Authors: Yassai Maryam , Gorski Jack
Published: April 2014

Thymocyte maturation consists of a number of stages, the goal of which is the production of functioning T cells that respond to foreign antigenic peptides using their clonotypic receptors. Selection of a productively rearranged TCR β-chain is the first stage in the process and occurs at the double-negative to double-positive (DP) transition. Later maturation stages are based on changes in markers such as CD5, CD69, or IL-7R. A stage in which α-chains are selected has also been identified using β-chain transgenic mice. Here we identify two additional selection stages in human thymocytes based on characteristics of the TCR. α selection is measured directly by identification of in-frame rearrangements and is associated with the appearance of CD3 on the DP thymocyte surface. The next stage has not yet been described and involves selection of thymocytes that express shorter TCR β-chain complementarity-determining region 3 (CDR3). This stage is associated with the acquisition of high levels of CDR3 by DP cells and the transition to SP thymocytes. The extent of CDR3 length selection observed is a function of the TCR V and J genes. We propose that CDR3 length selection is based on recognition of the MHC. Thus, there exist limitations on the allowable length of that portion of the TCR most intimately in contact with MHC and peptide. This may be a physical representation of positive selection.
Activation of Toll-Like Receptor 2 on Human Dendritic Cells Triggers Induction of IL-12, But Not IL-10
Authors: Thoma-Uszynski Sybille , Kiertscher Sylvia M. , Ochoa Maria Teresa , Bouis Deborah A. , Norgard Michael V. , Miyake Kensuke , Godowski Paul J. , Roth Michael D. , Modlin Robert L.
Published: April 2014

Mammalian Toll-like receptors (TLRs) are required for cell activation by bacterial lipoproteins (bLP) and LPS. Stimulation of monocytes with bLP and LPS results in a TLR-dependent induction of immunomodulatory genes leading to the production of pro-inflammatory cytokines. In this paper, we compared the expression and response of TLRs on monocytes and dendritic cells (DC). TLR2, but not TLR4, was detected on peripheral blood monocytes and DC, in lymphoid tissue CD1a+ DC as well as on in vitro monocyte-derived DC. Upon stimulation with bLP or LPS, monocytes produced IL-12 and IL-10 at similar levels, whereas monocyte-derived DC produced comparable levels of IL-12, but little IL-10. Greater than 90% of the bLP-induced production of IL-12 was blocked by anti-TLR2 mAb. Thus, DC express TLR2 and activation of this receptor by bLP provides an innate mechanism by which microbial pathogens preferentially activate cell-mediated immunity.
CD28 Utilizes Vav-1 to Enhance TCR-Proximal Signaling and NF-AT Activation
Authors: Michel Frédérique , Mangino Giorgio , Attal-Bonnefoy Géraldine , Tuosto Loretta , Alcover Andrés , Roumier Anne , Olive Daniel , Acuto Oreste
Published: April 2014

The mechanism through which CD28 costimulation potentiates TCR-driven gene expression is still not clearly defined. Vav-1, an exchange factor for Rho GTPases thought to regulate, mainly through Rac-1, various signaling components leading to cytokine gene expression, is tyrosine phosphorylated upon CD28 engagement. Here, we provide evidence for a key role of Vav-1 in CD28-mediated signaling. Overexpression of Vav-1 in Jurkat cells in combination with CD28 ligation strongly reduced the concentration of staphylococcus enterotoxin E/MHC required for TCR-induced NF-AT activation. Surprisingly, upon Vav-1 overexpression CD28 ligation sufficed to activate NF-AT in the absence of TCR engagement. This effect was not mediated by overexpression of ZAP-70 nor of SLP-76 but necessitated the intracellular tail of CD28, the intactness of the TCR-proximal signaling cascade, the Src-homology domain 2 (SH2) domain of Vav-1, and SLP-76 phosphorylation, an event which was favored by Vav-1 itself. Cells overexpressing Vav-1 formed lamellipodia and microspikes reminiscent of Rac-1 and Cdc42 activation, respectively, for which the SH2 domain of Vav-1 was dispensable. Together, these data suggest that CD28 engagement activates Vav-1 to boost TCR signals through a synergistic cooperation between Vav-1 and SLP-76 and probably via cortical actin changes to facilitate the organization of a signaling zone.
Conformational Epitopes on the Diabetes Autoantigen GAD65 Identified by Peptide Phage Display and Molecular Modeling
Authors: Myers Mark A. , Davies Janet M. , Tong Jonathan C. , Whisstock James , Scealy Marita , Mackay Ian R. , Rowley Merrill J.
Published: April 2014

The major diabetes autoantigen, glutamic acid decarboxylase (GAD65), contains a region of sequence similarity, including six identical residues PEVKEK, to the P2C protein of coxsackie B virus, suggesting that cross-reactivity between coxsackie B virus and GAD65 can initiate autoimmune diabetes. We used the human islet cell mAbs MICA3 and MICA4 to identify the Ab epitopes of GAD65 by screening phage-displayed random peptide libraries. The identified peptide sequences could be mapped to a homology model of the pyridoxal phosphate (PLP) binding domain of GAD65. For MICA3, a surface loop containing the sequence PEVKEK and two adjacent exposed helixes were identified in the PLP binding domain as well as a region of the C terminus of GAD65 that has previously been identified as critical for MICA3 binding. To confirm that the loop containing the PEVKEK sequence contributes to the MICA3 epitope, this loop was deleted by mutagenesis. This reduced binding of MICA3 by 70%. Peptide sequences selected using MICA4 were rich in basic or hydroxyl-containing amino acids, and the surface of the GAD65 PLP-binding domain surrounding Lys358, which is known to be critical for MICA4 binding, was likewise rich in these amino acids. Also, the two phage most reactive with MICA4 encoded the motif VALxG, and the reverse of this sequence, LAV, was located in this same region. Thus, we have defined the MICA3 and MICA4 epitopes on GAD65 using the combination of phage display, molecular modeling, and mutagenesis and have provided compelling evidence for the involvement of the PEVKEK loop in the MICA3 epitope.
Mechanism of IL-4-Mediated Up-Regulation of the Polymeric Ig Receptor: Role of STAT6 in Cell Type-Specific Delayed Transcriptional Response
Authors: Schjerven Hilde , Brandtzaeg Per , Johansen Finn-Eirik
Published: April 2014

The polymeric IgR (pIgR) mediates transport of dimeric IgA and pentameric IgM across mucosal epithelia, thereby generating secretory Abs. Its expression is up-regulated at the transcriptional level by IL-4 in HT-29 cells. In this study, we demonstrate that IL-4 mediates up-regulation of human pIgR through a 554-bp IL-4-responsive enhancer in intron 1. Mutation of a binding site for STAT-6 within this region abolished IL-4-induced enhancement, while an adjacent putative C/EBP site was dispensable. IL-4 treatment induced binding of STAT6 to the intronic STAT6 site, but cooperation with nearby upstream and downstream DNA elements was required for IL-4 responsiveness. Furthermore, IL-4-mediated increased transcription of the pIgR-derived enhancer, like the endogenous pIgR gene, required de novo protein synthesis. Interestingly, a conditionally active form of STAT6 sufficed to activate a pIgR-derived enhancer in HT-29 cells, but not in Cos-1 cells, suggesting a requirement for cell type-specific factors. Thus, STAT6 activation mediates a delayed transcriptional enhancement of pIgR by induction of a de novo synthesized protein that cooperates with STAT6 itself bound to its cognate DNA element in intron 1. This mechanism may represent a general strategy for how pleiotropic cytokines elicit cell type-specific transcriptional responses.
Cyclooxygenase-2 Expression in Macrophages: Modulation by Protein Kinase C-α
Authors: Giroux Mélanie , Descoteaux Albert
Published: April 2014

Cyclooxygenase-2 (COX-2) is an inducible enzyme responsible for high levels of PG production during inflammation and immune responses. Previous studies with pharmacological inhibitors suggested a role for protein kinase C (PKC) in PG production possibly by regulating COX-2 expression. In this study, we addressed the role of PKC-α in the modulation of COX-2 expression and PGE2 synthesis by the overexpressing of a dominant-negative (DN) mutant of this isoenzyme in the mouse macrophage cell line RAW 264.7. We investigated the effect of various stimuli on COX-2 expression, namely, LPS, IFN-γ, and the intracellular parasite Leishmania donovani. Whereas LPS-induced COX-2 mRNA and protein expression were down-regulated in DN PKC-α-overexpressing clones, IFN-γ-induced COX-2 expression was up-regulated in DN PKC-α-overexpressing clones with respect to normal RAW 264.7 cells. Measurements of PGE2 levels revealed a strong correlation between PGE2 secretion and IFN-γ-induced COX-2 mRNA and protein levels in DN PKC-α-overexpressing clones. Taken together, these results suggest a role for PKC-α in the modulation of LPS- and IFN-γ-induced COX-2 expression, as well as in IFN-γ-induced PGE2 secretion.
Activated Murine Endothelial Cells Have Reduced Immunogenicity for CD8+ T Cells: A Mechanism of Immunoregulation?
Authors: Marelli-Berg Federica M. , Scott Diane , Bartok Istvan , Peek Emma , Dyson Julian , Lechler Robert I.
Published: April 2014

The immunogenic properties of primary cultures of murine lung microvascular endothelial cells (EC) were analyzed. Resting endothelial cells were found to constitutively express low levels of MHC class I and CD80 molecules. IFN-γ treatment of EC resulted in a marked up-regulation of MHC class I, but no change was observed in the level of CD80 expression. No CD86 molecules were detectable under either condition. The ability of peptide-pulsed EC to induce the proliferation of either the HY-specific, H2-Kk-restricted CD8+ T cell clone (C6) or C6 TCR-transgenic naive CD8+ T cells was analyzed. Resting T cells were stimulated to divide by quiescent peptide-prepulsed EC, while peptide-pulsed, cytokine-activated EC lost the ability to induce T cell division. Furthermore, Ag presentation by cytokine-activated EC induced CD8+ T cell hyporesponsiveness. The immunogenicity of activated EC could be restored by adding nonsaturating concentrations of anti-H2-Kk Ab in the presence of an optimal concentration of cognate peptide. This is consistent with the suggestion that the ratio of TCR engagement to costimulation determines the outcome of T cell recognition. In contrast, activated peptide-pulsed EC were killed more efficiently by fully differentiated effector CD8+ T cells. Finally, evidence is provided that Ag recognition of EC can profoundly affect the transendothelial migration of CD8+ T cells. Taken together, these results suggest that EC immunogenicity is regulated in a manner that contributes to peripheral tolerance.
A Physiological Ligand of Positive Selection Is Recognized as a Weak Agonist
Authors: Berg Rance E. , Irion Stefan , Kattman Steve , Princiotta Michael F. , Staerz Uwe D.
Published: April 2014

Positive selection is a process that ensures that peripheral T cells express TCR that are self-MHC restricted. This process occurs in the thymus and requires both self-MHC and self-peptides. We have recently established a TCR transgenic (TCRtrans+) mouse model using the C10.4 TCR restricted to the MHC class Ib molecule, H2-M3. Having defined H2-M3 as the positively selecting MHC molecule, the severely limited number of H2-M3 binding peptides allowed us to characterize a mitochondrial NADH dehydrogenase subunit 1-derived 9-mer peptide as the physiological ligand of positive selection. Here, we demonstrate that the NADH dehydrogenase subunit 1 self-peptide is seen by mature C10.4 TCRtrans+ T cells as a weak agonist and induces positive selection at a defined concentration range. We also found that the full-length cognate peptide, a strong agonist for mature C10.4 TCRtrans+ T cells, initiated positive selection, albeit at significantly lower concentrations. At increased peptide concentrations, and thus increased epitope densities, either peptide only induced the development of partially functional T cells. We conclude that successful positive selection only proceeded at a defined, yet fairly narrow window of avidity.
Depletion of CD4 and CD8 T Lymphocytes in Mice In Vivo Enhances 1,25-Dihydroxyvitamin D3-Stimulated Osteoclast-Like Cell Formation In Vitro by a Mechanism That Is Dependent on Prostaglandin Synthesis
Authors: Grčević Danka , Lee Sun-Kyeong , Marušić Ana , Lorenzo Joseph A.
Published: April 2014

To investigate the role of T lymphocytes in osteoclastogenesis, we performed in vivo depletion of CD4 and/or CD8 T lymphocyte subsets and evaluated in vitro osteoclast-like cell (OCL) formation. T lymphocyte depletion (TLD) with mAbs was confirmed 24 h later by flow cytometry. OCL formation was stimulated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) in bone marrow and with recombinant mouse (rm) receptor activator of NF-κB ligand (RANK-L) and rmM-CSF in bone marrow and spleen cell cultures. OCL formation was up to 2-fold greater in 1,25-(OH)2D3-stimulated bone marrow cultures from TLD mice than in those from intact mice. In contrast, TLD did not alter OCL formation in bone marrow or spleen cell cultures that were stimulated with rmRANK-L and rmM-CSF. The effects of TLD seemed to be mediated by enhanced PG synthesis, because the PGE2 concentration in the medium of 1,25-(OH)2D3-stimulated bone marrow cultures from TLD mice was 5-fold higher than that in cultures from intact mice, and indomethacin treatment abolished the stimulatory effect of TLD on OCL formation. There was a 2-fold increase in RANK-L expression and an almost complete suppression of osteoprotegerin expression in 1,25-(OH)2D3-stimulated bone marrow cultures from TLD mice compared with those from intact mice. Although there was a small (20%) increase in IL-1α expression in 1,25-(OH)2D3-stimulated bone marrow cultures from TLD mice, TLD in mice lacking type I IL-1R and wild-type mice produced similar effects on OCL formation. Our data demonstrate that TLD up-regulates OCL formation in vitro by increasing PG production, which, in turn, produces reciprocal changes in RANK-L and osteoprotegerin expression. These results suggest that T lymphocytes influence osteoclastogenesis by altering bone marrow stromal cell function.
Endogenous Production of TGF-β Is Essential for Osteoclastogenesis Induced by a Combination of Receptor Activator of NF-κB Ligand and Macrophage-Colony- Stimulating Factor
Authors: Kaneda Toshio , Nojima Takaki , Nakagawa Mari , Ogasawara Aichi , Kaneko Hironori , Sato Takuya , Mano Hiroshi , Kumegawa Masayoshi , Hakeda Yoshiyuki
Published: April 2014

Differentiation of osteoclasts, the cells primarily responsible for bone resorption, is controlled by a variety of osteotropic hormones and cytokines. Of these factors, receptor activator of NF-κB (RANK) ligand (RANKL) has been recently cloned as an essential inducer of osteoclastogenesis in the presence of M-CSF. Here, we isolated a stroma-free population of monocyte/macrophage (M/Mφ)-like hemopoietic cells from mouse unfractionated bone cells that were capable of differentiating into mature osteoclasts by treatment with soluble RANKL (sRANKL) and M-CSF. However, the efficiency of osteoclast formation was low, suggesting the requirement for additional factors. The isolated M/Mφ-like hemopoietic cells expressed TGF-β and type I and II receptors of TGF-β. Therefore, we examined the effect of TGF-β on osteoclastogenesis. TGF-β with a combination of sRANKL and M-CSF promoted the differentiation of nearly all M/Mφ-like hemopoietic cells into cells of the osteoclast lineage. Neutralizing anti-TGF-β Ab abrogated the osteoclast generation. These TGF-β effects were also observed in cultures of unfractionated bone cells, and anti-TGF-β blocked the stimulatory effect of 1,25-dihydroxyvitamin D3. Translocation of NF-κB into nuclei induced by sRANKL in TGF-β-pretreated M/Mφ-like hemopoietic cells was greater than that in untreated cells, whereas TGF-β did not up-regulate the expression of RANK, the receptor of RANKL. Our findings suggest that TGF-β is an essential autocrine factor for osteoclastogenesis.
IL-2Rβ Agonist P1–30 Acts in Synergy with IL-2, IL-4, IL-9, and IL-15: Biological and Molecular Effects
Authors: Eckenberg Ralph , Moreau Jean-Louis , Melnyk Oleg , Thèze Jacques
Published: April 2014

From the sequence of human IL-2 we have recently characterized a peptide (p1–30), which is the first IL-2 mimetic described. P1–30 covers the entire α helix A of IL-2 and spontaneously folds into a α helical homotetramer mimicking the quaternary structure of a hemopoietin. This neocytokine interacts with a previously undescribed dimeric form of the human IL-2 receptor β-chain likely to form the p1–30 receptor (p1–30R). P1–30 acts as a specific IL-2Rβ agonist, selectively inducing activation of CD8 and NK lymphocytes. From human PBMC we have also shown that p1–30 induces the activation of lymphokine-activated killer cells and the production of IFN-γ. Here we demonstrate the ability of p1–30 to act in synergy with IL-2, -4, -9, and -15. These synergistic effects were analyzed at the functional level by using TS1β, a murine T cell line endogenously expressing the common cytokine γ gene and transfected with the human IL-2Rβ gene. At the receptor level, we show that expression of human IL-2Rβ is absolutely required to obtain synergistic effects, whereas IL-2Rα specifically impedes the synergistic effects obtained with IL-2. The results suggest that overexpression of IL-2Rα inhibits p1–30R formation in the presence of IL-2. Finally, concerning the molecular effects, although p1–30 alone induces the antiapoptotic molecule bcl-2, we show that it does not influence mRNA expression of c-myc, c-jun, and c-fos oncogenes. In contrast, p1–30 enhances IL-2-driven expression of these oncogenes. Our data suggest that p1–30R (IL-2Rβ)2 and intermediate affinity IL-2R (IL-2Rβγ), when simultaneously expressed at the cell surface, may induce complementary signal transduction pathways and act in synergy.
Maturation-Dependent Expression and Function of the CD49d Integrin on Monocyte-Derived Human Dendritic Cells
Authors: Puig-Kröger Amaya , Sanz-Rodríguez Francisco , Longo Natividad , Sánchez-Mateos Paloma , Botella Luisa , Teixidó Joaquín , Bernabéu Carmelo , Corbí Angel L.
Published: April 2014

Dendritic cells (DC) are highly specialized APC that are critical for the initiation of T cell-dependent immune responses. DC exert a sentinel function while immature and, after activation by inflammatory stimuli or infectious agents, mature and migrate into lymphoid organs to prime T cells. We have analyzed integrin expression on monocyte-derived DC (MDDC) and found that expression of CD49d integrins (CD49d/CD29 and CD49d/β7) was induced/up-regulated during TNF-α- or LPS-initiated MDDC maturation, reflecting the induction/up-regulation of CD49d and β7 mRNA. CD49d mRNA steady-state level increased more than 10 times during maturation, with the highest levels observed 24 h after TNF-α treatment. CD49d integrin expression conferred mature MDDC with an elevated capacity to adhere to the CS-1 fragment of fibronectin, and also mediated transendothelial migration of mature MDDC. Up-regulation of CD49d integrin expression closely paralleled that of the mature DC marker CD83. CD49d integrin expression was dependent on cell maturation, as its induction was abrogated by N-acetylcysteine, which inhibits NF-κB activation and the functional and phenotypic maturation of MDDC. Moreover, CD49d integrin up-regulation and MDDC maturation were prevented by SB203580, a specific inhibitor of p38 mitogen-activated protein kinase, but were almost unaffected by the mitogen-activated protein/extracellular signal-related kinase kinase 1/2 inhibitor PD98059. Our results support the existence of a link between functional and phenotypic maturation of MDDC and CD49d integrin expression, thus establishing CD49d as a maturation marker for MDDC. The differential expression of CD49d on immature and mature MDDC might contribute to their distinct motility capabilities and mediate mature DC migration into lymphoid organs.
Fas/Fas Ligand Interactions Promote Activation-Induced Cell Death of NK T Lymphocytes
Authors: Leite-de-Moraes Maria C. , Herbelin André , Gouarin Christine , Koezuka Yasuhiko , Schneider Elke , Dy Michel
Published: April 2014

NKT cells are a versatile population whose immunoregulatory functions are modulated by their microenvironment. We demonstrate herein that in addition to their IFN-γ production, NKT lymphocytes stimulated with IL-12 plus IL-18 in vitro underwent activation in terms of CD69 expression, blast transformation, and proliferation. Yet they were unable to survive in culture because, once activated, they were rapidly eliminated by apoptosis, even in the presence of their survival factor IL-7. This process was preceded by up-regulation of Fas (CD95) and Fas ligand expression in response to IL-12 plus IL-18 and was blocked by zVAD, a large spectrum caspase inhibitor, as well as by anti-Fas ligand mAb, suggesting the involvement of the Fas pathway. In accordance with this idea, NKT cells from Fas-deficient C57BL/6-lpr/lpr mice did not die in these conditions, although they shared the same features of cell activation as their wild-type counterpart. Activation-induced cell death occurred also after TCR engagement in vivo, since NKT cells became apoptotic after injection of their cognate ligand, α-galactosylceramide, in wild-type, but not in Fas-deficient, mice. Taken together, our data provide the first evidence for a new Fas-dependent mechanism allowing the elimination of TCR-dependent or -independent activated NKT cells, which are potentially dangerous to the organism.
CXCR4 Receptor Expression on Human Retinal Pigment Epithelial Cells from the Blood-Retina Barrier Leads to Chemokine Secretion and Migration in Response to Stromal Cell-Derived Factor 1α
Authors: Crane Isabel J. , Wallace Carol A. , McKillop-Smith Susan , Forrester John V.
Published: April 2014

Retinal pigment epithelial (RPE) cells form part of the blood-retina barrier and have recently been shown to produce various chemokines in response to proinflammatory cytokines. As the scope of chemokine action has been shown to extend beyond the regulation of leukocyte migration, we have investigated the expression of chemokine receptors on RPE cells to determine whether they could be a target for chemokine signaling. RT-PCR analysis indicated that the predominant receptor expressed on RPE cells was CXCR4. The level of CXCR4 mRNA expression, but not cell surface expression, increased on stimulation with IL-1β or TNF-α. CXCR4 protein could be detected on the surface of 16% of the RPE cells using flow cytometry. Calcium mobilization in response to the CXCR4 ligand stromal cell-derived factor 1α (SDF-1α) indicated that the CXCR4 receptors were functional. Incubation with SDF-1α resulted in secretion of monocyte chemoattractant protein-1, IL-8, and growth-related oncogene α. RPE cells also migrated in response to SDF-1α. As SDF-1α expression by RPE cells was detected constitutively, we postulate that SDF-1–CXCR4 interactions may modulate the affects of chronic inflammation and subretinal neovascularization at the RPE site of the blood-retina barrier.
Identification of a Complex that Binds to the CD154 3′ Untranslated Region: Implications for a Role in Message Stability During T Cell Activation
Authors: Barnhart Bryan , Kosinski Penelope A. , Wang Zuoren , Ford Gregory S. , Kiledjian Megerditch , Covey Lori R.
Published: April 2014

CD154 expression is regulated throughout a time course of CD3-dependent T cell activation by differential mRNA decay. To understand the molecular basis of the “stability” phase of this pathway, experiments were conducted to identify sequences and specific complexes important in this regulation. Gel retardation assays using extracts from both Jurkat T cells and CD3-activated CD4+ T cells revealed a major complex (complex I) that bound a 65-bp highly CU-rich region of the CD154 3′ untranslated region. The specificity of the CU-rich element for complex-I formation was confirmed by disruption of this complex by oligo(dCT) competition. Formation of complex I strongly correlated with CD154 mRNA stability across a time course of T cell activation. UV cross-linking identified a major oligo(dCT)-sensitive species at ∼90 kDa that showed induced and increased expression in extracts from 24- and 48-hr anti-CD3-activated T cells, respectively. This protein was absent in equivalent extracts from resting or 2-h-activated T cells. Using an in vitro decay assay, we found that a CD154-specific transcript was more rapidly degraded in 2-h-activated extract and stabilized in the 24- and 48-h extracts compared to extracts from resting T cells. Disruption of complex I resulted in the rapid decay of a CD154-specific transcript demonstrating a functional role for complex I in mRNA stabilization in vitro. These studies support a model of posttranscriptional regulation of CD154 expression being controlled in part by the interaction of a poly(CU)-binding complex with a specific sequence in the 3′ untranslated region.
Molecular Recognition of Human CD1b Antigen Complexes: Evidence for a Common Pattern of Interaction with αβ TCRs
Authors: Melián Agustín , Watts Gerald F. M. , Shamshiev Abdijapar , De Libero Gennaro , Clatworthy Anne , Vincent Michael , Brenner Michael B. , Behar Samuel , Niazi Kayvan , Modlin Robert L. , Almo Steven , Ostrov David , Nathenson Stanley G. , Porcelli Steven A.
Published: April 2014

Ag-specific T cell recognition is mediated through direct interaction of clonotypic TCRs with complexes formed between Ag-presenting molecules and their bound ligands. Although characterized in substantial detail for class I and class II MHC encoded molecules, the molecular interactions responsible for TCR recognition of the CD1 lipid and glycolipid Ag-presenting molecules are not yet well understood. Using a panel of epitope-specific Abs and site-specific mutants of the CD1b molecule, we showed that TCR interactions occur on the membrane distal aspects of the CD1b molecule over the α1 and α2 domain helices. The location of residues on CD1b important for this interaction suggested that TCRs bind in a diagonal orientation relative to the longitudinal axes of the α helices. The data point to a model in which TCR interaction extends over the opening of the putative Ag-binding groove, making multiple direct contacts with both α helices and bound Ag. Although reminiscent of TCR interaction with MHC class I, our data also pointed to significant differences between the TCR interactions with CD1 and MHC encoded Ag-presenting molecules, indicating that Ag receptor binding must be modified to accommodate the unique molecular structure of the CD1b molecule and the unusual Ags it presents.
Adenovirus E1A Oncogene Expression in Tumor Cells Enhances Killing by TNF-Related Apoptosis-Inducing Ligand (TRAIL)
Authors: Routes John M. , Ryan Sharon , Clase Amanda , Miura Tanya , Kuhl Alicia , Potter Terry A. , Cook James L.
Published: April 2014

Expression of the adenovirus serotype 5 (Ad5) E1A oncogene sensitizes cells to apoptosis by TNF-α and Fas-ligand. Because TNF-related apoptosis-inducing ligand (TRAIL) kills cells in a similar manner as TNF-α and Fas ligand, we asked whether E1A expression might sensitize cells to lysis by TRAIL. To test this hypothesis, we examined TRAIL-induced killing of human melanoma (A2058) or fibrosarcoma (H4) cells that expressed E1A following either infection with Ad5 or stable transfection with Ad5-E1A. E1A-transfected A2058 (A2058-E1A) or H4 (H4-E1A) cells were highly sensitive to TRAIL-induced killing, but Ad5-infected cells expressing equally high levels of E1A protein remained resistant to TRAIL. Infection of A2058-E1A cells with Ad5 reduced their sensitivity to TRAIL-dependent killing. Therefore, viral gene products expressed following infection with Ad5 inhibited the sensitivity to TRAIL-induced killing conferred by transfection with E1A. E1B and E3 gene products have been shown to inhibit TNF-α- and Fas-dependent killing. The effect of these gene products on TRAIL-dependent killing was examined by using Ad5-mutants that did not express either the E3 (H5dl327) or E1B-19K (H5dl250) coding regions. A2058 cells infected with H5dl327 were susceptible to TRAIL-dependent killing. Furthermore, TRAIL-dependent killing of A2058-E1A cells was not inhibited by infection with H5dl327. Infection with H5dl250 sensitized A2058 cells to TRAIL-induced killing, but considerably less than H5dl327-infection. In summary, expression of Ad5-E1A gene products sensitizes cells to TRAIL-dependent killing, whereas E3 gene products, and to a lesser extent E1B-19K, inhibit this effect.
Role of IL-5 in Innate and Adaptive Immunity to LarvalStrongyloides stercoralisin Mice
Authors: Herbert De’Broski R. , Lee James J. , Lee Nancy A. , Nolan Thomas J. , Schad Gerhard A. , Abraham David
Published: April 2014

Protective immunity to Strongyloides stercoralis infective larvae in mice has been shown to be dependent on IL-5 based on mAb depletion studies. The goal of this study was to determine the functional role of IL-5 during the innate and adaptive immune response to larval S. stercoralis in mice. In these studies, three strains of mice were used: wild-type C57BL/6J (WT), IL-5 knockout (KO), and IL-5 transgenic (TG). Innate responses to the larvae indicated that there was enhanced survival in the KO animals and decreased survival in the TG animals compared with WT. Furthermore, killing of larvae in TG mice was associated with eosinophil infiltration and degranulation. In studying the adaptive immune response, it was observed that immunization of KO mice did not lead to the development of protective immunity. Experiments were then performed to determine whether KO mice reconstituted with Abs or cells could then develop protective immunity. KO mice displayed protective immunity via a granulocyte-dependent mechanism following injection of purified IgM from immune wild-type animals. Immunity in KO mice could also be reconstituted by the injection of eosinophils at the time of immunization. These eosinophils did not participate in actively killing the challenge infection, but rather were responsible for the induction of a protective Ab response. We conclude that IL-5 is required in the protective immune response for the production of eosinophils, and that eosinophils were involved in larval killing during innate immunity and in the induction of protective Abs in the adaptive immune response.
CD47 Ligation Selectively Inhibits the Development of Human Naive T Cells into Th1 Effectors
Authors: Avice Marie-Noëlle , Rubio Manuel , Sergerie Martin , Delespesse Guy , Sarfati Marika
Published: April 2014

The CD47 Ag, also named integrin-associated protein, was recently reported to regulate the production of IL-12 by human monocytes and dendritic cells. The present study shows that CD47 ligation by CD47 mAb in primary cultures of cord blood mononuclear cells inhibits IL-12-driven Th1 cell development, as revealed by the cytokine secretion profile at restimulation and IFN-γ production at the single-cell level. F(ab′)2 fragments of CD47 mAb or the synthetic peptide 4N1K, corresponding to the CD47 binding site of thrombospondin, display the same activity. CD47 engagement does not change the phenotype of IL-12-primed cells from Th1 to Th2 or affect IL-4-induced Th2 cell development. Moreover, CD47 mAb inhibits IL-12- but not IL-4-induced IL-2 production as well as IFN-γ in primary cultures, which was correlated with a decrease of the IL-12Rβ2 chain expression. Inclusion of exogenous IL-2 at priming corrects IL-12R expression as well as the inhibition of Th1 cell development. The data thus underline the role of IL-2 in Th1 cell development and further suggest that targeting IL-2 and IL-12 simultaneously may have some therapeutic advantage in Th1 autoimmune diseases.