bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reduced Generation but Efficient TCRβ-Chain Selection of CD4+8+ Double-Positive Thymocytes in Mice with Compromised CD3 Complex Signaling
Authors: Würch Andreas , Biro Judit , Falk Ingrid , Mossmann Horst , Eichmann Klaus 
Published: December 2022

Abstract
Maturation to the CD4+8+ double-positive (DP) stage of thymocyte development is restricted to cells that have passed TCRβ selection, an important checkpoint at which immature CD4−8− double-negative (DN) cells that express TCRβ polypeptide chains are selected for further maturation. The generation of DP thymocytes following TCRβ selection is dependent on cellular survival, differentiation, and proliferation, and the entire process appears to be mediated by the pre-TCR/CD3 complex. In this study, we investigate the signaling requirements for TCRβ selection using mice single deficient and double deficient for CD3ζ/η and/or p56lck. While the numbers of DP cells are strongly reduced in the single-deficient mice, a further drastic reduction in the generation of DP thymocytes is seen in the double-deficient mice. The poor generation of DP cells in the mutant mice is primarily due to an impaired ability of CD25+ DN thymocytes to proliferate following expression of a TCRβ-chain. Nevertheless, the residual DP cells in all mutant mice are strictly selected for expression of TCRβ polypeptide chains. DN thymocytes of mutant mice expressed TCRβ and CD3ε at the cell surface and contained mRNA for pre-Tα, but not for clonotypic TCRα-chains, together suggesting that TCRβ selection is mediated by pre-TCR signaling in all cases. The data suggest differential requirements of pre-TCR signaling for cell survival on the one hand, and for the proliferative burst associated with TCRβ selection on the other.
Inhibition of Cell Cycle Progression by Rapamycin Induces T Cell Clonal Anergy Even in the Presence of Costimulation
Authors: Powell Jonathan D. , Lerner Cara G. , Schwartz Ronald H. 
Published: December 2022

AbstractCostimulation (signal 2) has been proposed to inhibit the induction of T cell clonal anergy by either directly antagonizing negative signals arising from TCR engagement (signal 1) or by synergizing with signal 1 to produce IL-2, which in turn leads to proliferation and dilution of negative regulatory factors. To better define the cellular events that lead to the induction of anergy, we used the immunosuppressive agent rapamycin, which blocks T cell proliferation in late G1 phase but does not affect costimulation-dependent IL-2 production. Our data demonstrate that full T cell activation (signal 1 plus 2) in the presence of rapamycin results in profound T cell anergy, despite the fact that these cells produce copious amounts of IL-2. Similar to conventional anergy (induction by signal 1 alone), the rapamycin-induced anergic cells show a decrease in mitogen-activated protein kinase activation, and these cells can be rescued by culture in IL-2. Interestingly, the rapamycin-induced anergic cells display a more profound block in IL-3 and IFN-γ production upon rechallenge. Finally, in contrast to rapamycin, full T cell activation in the presence of hydroxyurea (which inhibits the cell cycle in early S phase) did not result in anergy. These data suggest that it is neither the direct effect of costimulation nor the subsequent T cell proliferation that prevents anergy induction, but rather the biochemical events that occur upon progression through the cell cycle from G1 into S phase.
Role of a STAT Binding Site in the Regulation of the Human Perforin Promoter
Authors: Yu Cheng-Rong , Ortaldo John R. , Curiel Rafael E. , Young Howard A. , Anderson Stephen K. , Gosselin Pierre 
Published: December 2022

AbstractThe pore-forming protein perforin is preferentially expressed in NK and cytotoxic T cells. To investigate the molecular regulation of human perforin gene transcription, the activity of the human perforin promoter was analyzed in human NK and T cell lines using various promoter fragments linked to a luciferase reporter gene. A core promoter was identified within 55 bp upstream of the transcription start site. This promoter region contains a guanine/cytosine box and has basal activity in YT, Kit225-k6, and Jurkat cells. A strong enhancer activity was identified between positions −1136 and −1076, a region that includes a STAT-like element. This enhancer region was active in YT cells, which have constitutive perforin expression and activated STAT3 protein, but not in Kit225-k6 or Jurkat cells, which do not have constitutive perforin expression. Mutation of the STAT binding site resulted in a dramatic down-regulation of promoter activity. Electrophoretic mobility shift assays, using a probe containing the STAT element of the perforin promoter, indicated that this element can bind STAT3 from YT cells. Moreover, the STAT element was shown to bind STAT5a/b induced by IL-2 as well as STAT1α induced by IL-6 in human NK cells. Together, these results suggest that STAT proteins play a key role in perforin gene transcription and provide a model by which cytokines can regulate perforin gene expression.
Affinity Modulation of Very Late Antigen-5 Through Phosphatidylinositol 3-Kinase in Mast Cells
Authors: Kinashi Tatsuo , Asaoka Tetsuo , Setoguchi Ruri , Takatsu Kiyoshi 
Published: December 2022

AbstractAdhesiveness of integrins is up-regulated rapidly by a number of molecules, including growth factors, cytokines, chemokines, and other cell surface receptors, through a mechanism termed inside-out signaling. The inside-out signaling pathways are thought to alter integrin affinity for ligand, or cell surface distribution of integrin by diffusion/clustering. However, it remains to be clarified whether any physiologically relevant agonists induce a rapid change in the affinity of β1 integrins and how ligand-binding affinity is modulated upon stimulation. In this study, we reported that affinity of β1 integrin very late Ag-5 (VLA-5) for fibronectin was rapidly increased in bone marrow-derived mast cells by Ag cross-linking of FcεRI. Ligand-binding affinity of VLA-5 was also augmented by receptor tyrosine kinases when the phospholipase Cγ-1/protein kinase C pathway was inhibited. Wortmannin suppressed induction of the high affinity state VLA-5 in either case. Conversely, introduction of a constitutively active p110 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) increased the binding affinity for fibronectin. Failure of a constitutively active Akt to stimulate adhesion suggested that the affinity modulation mechanisms mediated by PI 3-kinase are distinct from the mechanisms to control growth and apoptosis by PI 3-kinase. Taken together, our findings demonstrated that the increase of affinity of VLA-5 was induced by physiologically relevant stimuli and PI 3-kinase was a critical affinity modulator of VLA-5.
Endogenous Platelet-Activating Factor Is Critically Involved in Effector Functions of Eosinophils Stimulated with IL-5 or IgG
Authors: Bartemes Kathleen R. , McKinney Shannon , Gleich Gerald J. , Kita Hirohito 
Published: December 2022

Abstract
Eosinophil activation and subsequent release of inflammatory mediators are implicated in the pathophysiology of allergic diseases. Eosinophils are activated by various classes of secretagogues, such as cytokines (e.g., IL-5), lipid mediators (e.g., platelet-activating factor (PAF)), and Ig (e.g., immobilized IgG). However, do these agonists act directly on eosinophils or indirectly through the generation of intermediate active metabolites? We now report that endogenous PAF produced by activated eosinophils plays a critical role in eosinophil functions. Human eosinophils produced superoxide when stimulated with immobilized IgG, soluble IL-5, or PAF. Pretreating eosinophils with pertussis toxin abolished their responses to these stimuli, suggesting involvement of a metabolite(s) that acts on G proteins. Indeed, PAF was detected in supernatants from eosinophils stimulated with IgG or IL-5. Furthermore, structurally distinct PAF antagonists, including CV6209, hexanolamine PAF, and Y-24180 (israpafant), inhibited IgG- or IL-5-induced superoxide production and degranulation. Previous reports indicated that exogenous PAF stimulates eosinophil eicosanoid production through formation of lipid bodies. We found in this study that IgG or IL-5 also induces lipid body formation and subsequent leukotriene C4 production mediated by endogenous PAF. Finally, inhibition of cytosolic phospholipase A2, one of the key enzymes involved in PAF synthesis, attenuated both PAF production and effector functions of eosinophils. These findings suggest that endogenous PAF plays important roles in eosinophil functional responses to various exogenous stimuli, such as cytokines and Igs. Therefore, inhibition of PAF synthesis or action may be beneficial for the treatment of eosinophilic inflammation.
Altered Helper T Lymphocyte Function Associated with Chronic Hepatitis B Virus Infection and its Role in Response to Therapeutic Vaccination in Humans
Authors: Livingston Brian D. , Alexander Jeff , Crimi Claire , Oseroff Carla , Celis Esteban , Daly Kieran , Guidotti Luca G. , Chisari Francis V. , Fikes John , Chesnut Robert W. , Sette Alessandro 
Published: December 2022

AbstractTheradigm-hepatitis B virus (HBV) is an experimental lipopeptide vaccine designed to stimulate induction of HBV-specific CTL responses in HLA-A2 individuals. Previous studies had demonstrated high immunogenicity in healthy volunteers, but comparatively weak CTL responses in chronically infected HBV patients. Herein, we examined helper T lymphocyte (HTL) responses in chronically infected patients. Despite normal proliferation and IL-2 secretion, IL-12 and IFN-γ secretion in vitro in response to the vaccine was reduced compared with healthy volunteers. A similar pattern of cytokine secretion was observed following mitogen stimulation, suggesting a general altered balance of Th1/Th2 responses. Further analysis indicated that HTL recall responses to whole tetanus toxoid protein were reduced in chronically infected subjects, and reduced responsiveness correlated with the outcome of Theradigm-HBV immunization. Finally, experiments in HBV transgenic mice indicated that the nonnatural Pan DR HTL epitope, PADRE, is capable of inducing high levels of IFN-γ secretion and that its inclusion in a lipopeptide incorporating an immunodominant Ld-restricted CTL epitope resulted in breaking tolerance at the CTL level. Overall, our results demonstrate an alteration in the quality of HTL responses induced in chronically infected HBV patients and suggest that use of a potent HTL epitope may be important to overcome CTL tolerance against specific HBV Ags.
Cutting Edge: Stable Epigenetic Inheritance of Regional IFN-γ Promoter Demethylation in CD44highCD8+ T Lymphocytes
Authors: Fitzpatrick David R. , Shirley Kym M. , Kelso Anne 
Published: December 2022

AbstractGenomic DNA methylation patterns influence the development and maintenance of function during cellular differentiation. Methylation of regulatory sequences can have long-lasting effects on gene expression if inherited in an epigenetic manner. Recent work suggests that DNA methylation has a regulatory role in differential cytokine gene expression in primary T lymphocytes. Here we show, by clonal lineage analysis, that methylation patterns in the IFN-γ promoter exhibit long term faithful inheritance in CD44highCD8+ T cells and their progeny, through 16 cell divisions and a clonal expansion of 5 orders of magnitude. Moreover, the demethylated IFN-γ promoter is faithfully inherited following the withdrawal of T cell stimulation and the loss of detectable IFN-γ mRNA, consistent with passive rather than active maintenance mechanisms. This represents a form of stable cellular memory, of defined epigenetic characteristics, that may contribute to the maintenance of T cell cytokine expression patterns and T cell memory.
Suppression of Antigen-Specific Th2 Cell-Dependent IgM and IgG1 Production Following Norepinephrine Depletion In Vivo
Authors: Kohm A. P. , Sanders V. M. 
Published: December 2022

AbstractThe mechanism by which the Th2 cell-dependent Ab response is modulated by the sympathetic neurotransmitter norepinephrine (NE) was investigated. Our model system used the severe combined immunodeficient (scid) mouse that was depleted of NE with 6-hydroxydopamine before reconstitution with a clone of β2-adrenergic receptor (β2AR)neg KLH-specific Th2 cells and resting trinitrophenyl (TNP)-specific β2ARpos B cells enriched from the spleens of unimmunized mice. Following challenge with TNP-keyhole limpet hemocyanin (KLH), Ab production in these mice was hapten-, carrier-, and allotype-specific as well as MHC restricted. Depletion of NE resulted in a 50–75% suppression of the primary anti-TNP IgM response compared with that of NE-intact controls, while the secondary IgM response returned to control levels. In contrast, both the primary and secondary anti-TNP IgG1 responses were suppressed by 85 and 40%, respectively. Using NE-intact mice exposed to either a βAR- or αAR-selective antagonist, the effect of NE on the Ab response was shown to be mediated by the βAR. In addition, administration of a β2AR-selective agonist to NE-depleted mice partially reversed the suppressed Ab response that resulted from NE depletion. Expression of the β2AR on TNP-specific B cells was confirmed by radioligand binding, immunofluorescence, and cAMP analysis. Also, while splenic histology was comparable in NE-intact and NE-depleted mice before Ag exposure, follicle expansion and germinal center formation were suppressed in NE-depleted mice after Ag exposure. Taken together, these results suggest that NE stimulation of the β2AR expressed on B cells is necessary for the maintenance of an optimal primary and secondary Th2 cell-dependent Ab response in vivo.
HIV-1 Nef Expression Inhibits the Activity of a Ca2+-Dependent K+ Channel Involved in the Control of the Resting Potential in CEM Lymphocytes
Authors: Zegarra-Moran Olga , Rasola Andrea , Rugolo Michela , Porcelli Anna M. , Rossi Bernard , Galietta Luis J. V. 
Published: December 2022

AbstractThe HIV-1 Nef protein plays an important role in the development of the pathology associated with AIDS. Despite various studies that have dealt with different aspects of Nef function, the complete mechanism by which it alters the physiology of infected cells remains to be established. Nef can associate with cell membranes, therefore supporting the hypothesis that it might interact with membrane proteins as ionic channels and modify their electrical properties. By using the patch-clamp technique, we found that Nef expression determines a 25-mV depolarization of lymphoblastoid CEM cells. Both charybdotoxin (CTX) and the membrane-permeable Ca2+ chelator BAPTA/AM depolarized the membrane of native cells without modifying that of Nef-transfected cells. These data suggested that the resting potential in native CEM cells is settled by a CTX- and Ca2+-sensitive K+ channel (KCa,CTX), whose activity is absent in Nef-expressing cells. This was confirmed by direct measurements of whole-cell KCa,CTX currents. Single-channel recordings on excised patches showed that a KCa,CTX channel of 35 pS with a half-activation near 400 nM Ca2+ was present in both native and Nef-transfected cells. The measurements of free intracellular Ca2+ were not different in the two cell lines, but Nef-transfected cells displayed an increased Ca2+ content in ionomycin-sensitive stores. Taken together, these results indicate that Nef expression alters the resting membrane potential of the T lymphocyte cell line by inhibiting a KCa,CTX channel, possibly by intervening in the regulation of intracellular Ca2+ homeostasis.
Chronic Inflammatory Disease Alters Adhesion Molecule Requirements for Acute Neutrophil Emigration in Mouse Skin
Authors: Mizgerd Joseph P. , Bullard Daniel C. , Hicks M. John , Beaudet Arthur L. , Doerschuk Claire M. 
Published: December 2022

Abstract
Mutant mice triply deficient in ICAM-1, E-selectin, and P-selectin did not develop the neutrophilic skin lesions that spontaneously arise in mutants doubly deficient in E-selectin and P-selectin. Thus, ICAM-1 is essential to skin disease resulting from endothelial selectin deficiency. During experimental dermatitis, acute neutrophil emigration was completely prevented in young mice deficient in both selectins (E/P and E/P/I mutants). However, older E/P mutants with spontaneous skin lesions displayed an endothelial selectin-independent pathway for acute neutrophil emigration. In contrast, emigration remained compromised in E/P/I mutants and CD18 mutants regardless of age or lesions. Experimentally induced chronic lesions elicited this pathway for acute emigration in young E/P mutants. Thus, an endothelial selectin-independent pathway for acute neutrophil emigration is induced in E/P mice by chronic inflammation at distant sites, and this pathway may contribute to skin disease resulting from endothelial selectin deficiency.
Lungkine, a Novel CXC Chemokine, Specifically Expressed by Lung Bronchoepithelial Cells
Authors: Rossi Devora L. , Hurst Stephen D. , Xu Yuming , Wang Wei , Menon Satish , Coffman Robert L. , Zlotnik Albert 
Published: December 2022

AbstractWe describe a novel mouse CXC chemokine that is selectively expressed in lung epithelial cells and up-regulated in various lung inflammation models. Although this chemokine clusters with other ELR-CXC chemokines, none of them can confidently be assigned to be its human homologue based on sequence identity. In addition, the highly restricted mRNA tissue distribution of this chemokine differentiates it from all previously described chemokines: Lungkine could not be detected in any of the 70 cDNA libraries analyzed corresponding to specific murine cell populations and tissues. High levels of Lungkine mRNA were specifically detected in the lung and at lower levels in fetal lung tissue by Northern blot and in situ hybridization, suggesting a potential role for this chemokine during lung development. Moreover, Lungkine protein is secreted into the airway spaces and induces the in vitro and in vivo migration of neutrophils, suggesting that it is involved in lung-specific neutrophil trafficking. Using fluorescent in situ hybridization, we show that Lungkine maps to mouse chromosome 5.
Impaired T Cell Proliferation in Acute Dengue Infection
Authors: Mathew Anuja , Kurane Ichiro , Green Sharone , Vaughn David W. , Kalayanarooj Siripen , Suntayakorn Saroj , Ennis Francis A. , Rothman Alan L. 
Published: December 2022

AbstractDecreased proliferative responses to mitogens and recall Ags have been observed in PBMC obtained during several acute human viral infections. To determine whether cell-mediated responses are altered during acute dengue infection, we examined the proliferative responses of PBMC from children enrolled in a prospective study of dengue infections in Thailand. All responses of PBMC during acute illness were compared with the same patients’ PBMC obtained at least 6 mo after their infection. Proliferative responses to PHA, anti-CD3, tetanus toxoid, and dengue Ags were decreased significantly in PBMC obtained during the acute infection. The proliferative responses to PHA were restored by the addition of gamma-irradiated autologous convalescent or allogeneic PBMC. Cell contact with the irradiated PBMC was necessary to restore proliferation. Non-T cells from the acute PBMC of dengue patients did not support proliferation of T cells from control donors in response to PHA, but T cells from the PBMC of patients with acute dengue proliferated if accessory cells from a control donor were present. Addition of anti-CD28 Abs restored anti-CD3-induced proliferation of the PBMC of some patients. The percentage of monocytes was reduced in the acute sample of PBMC of the dengue patients. Addition of IL-2 or IL-7, but not IL-4 or IL-12, also restored proliferation of acute PBMC stimulated with anti-CD3. The results demonstrate that both quantitative and qualitative defects in the accessory cell population during acute dengue illness result in a depression of in vitro T cell proliferation.
Sensitization of AIDS-Kaposi’s Sarcoma Cells to Apo-2 Ligand-Induced Apoptosis by Actinomycin D
Authors: Mori Shunsuke , Murakami-Mori Kaoru , Nakamura Shuji , Ashkenazi Avi , Bonavida Benjamin 
Published: December 2022

AbstractKaposi’s sarcoma (KS) is the most frequent malignancy associated with HIV infection (AIDS-KS), a complication that leads to high mortality and morbidity. AIDS-KS cells are resistant to killing by chemotherapeutic drugs/NK cells and Fas-induced apoptosis, suggesting that the acquisition of antiapoptotic characteristics by AIDS-KS cells may contribute to their prolonged survival. Apo-2 ligand (Apo-2L)/TNF-related apoptosis-inducing ligand, a new member of the TNF family, has been identified as an apoptosis-inducing molecule. In this study we examined the sensitivity of 10 different AIDS-KS isolates to Apo-2L-mediated cytotoxicity. AIDS-KS cells were relatively resistant to Apo-2L; however, Apo-2L and actinomycin D (Act D) used in combination synergistically potentiated the induction of cell death in nine of the 10 isolates. Apo-2L induced apoptosis in &gt;80% of AIDS-KS cells pretreated with Act D. The caspase inhibitors, zIETD-fmk and zDEVD-fmk, inhibited apoptosis in AIDS-KS by sApo-2L, suggesting that caspase 3-like and caspase 8 or 10 activities are essential for Apo-2L-mediated apoptosis. Act D treatment of AIDS-KS cells markedly and selectively down-regulated Bcl-xL expression, while the expressions of decoy receptors 1 and 2, Bax, cellular FLICE (Fas-associated death domain protein-like IL-1-converting enzyme) inhibitory protein, FADD (Fas-associated death domain protein), procaspase 8, and p53 were not affected. These findings suggest the possible involvement of Bcl-xL in Act D-induced sensitization of AIDS-KS cells to Apo-2L-mediated apoptosis. Furthermore, Act D did not sensitize PBMC or fibroblast cells to Apo-2L. Thus, Apo-2L and Act D used in combination may be of therapeutic value in the treatment of AIDS-KS.
Cutting Edge: Tumor Secreted Heat Shock-Fusion Protein Elicits CD8 Cells for Rejection
Authors: Yamazaki Koichi , Nguyen Timmy , Podack Eckhard R. 
Published: December 2022

AbstractThe endoplasmic reticulum resident heat shock protein gp96 chaperons peptides, including those derived from tumor Ags, on their way to presentation by MHC class I. Replacement of the endoplasmic reticulum retention signal of gp96 with the Fc portion of murine IgG1 generated a secretory form of gp96, gp96-Ig. Tumor cells secreting gp96-Ig exhibited decreased tumorigenicity and increased immunogenicity in vivo and were rejected after initial growth. Rejection required CD8 T cells during the priming and effector phase. CD4 T cells were not required for rejection in either phase. Carrageenan, a compound known to inactivate macrophages in vivo, did not diminish CD8-mediated tumor rejection. Therefore, immunization with tumors secreting gp96-Ig generates efficient tumor-rejecting CD8 CTL without requirement for CD4 or macrophage help. In contrast, immunization with purified, tumor-derived gp96 or with irradiated tumor cells requires both.