bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced Cytotoxic T Cell Activity in IL-4-Deficient Mice
Authors: Villacres Maria C. , Bergmann Cornelia C.
Published: December 2022

CD8+ effectors are critical components of type 1 responses against viral infections as well as for antiviral vaccines. IL-4 plays a clear role as an inhibitor of CD4+ Th1 cells; however, its role in CD8+ T cell regulation appears to be more complex. Thus, IL-4 may augment CD8+ T cell growth, but also limit effector function. Moreover, abundant IL-4 is inhibitory for viral clearance, but the lack of IL-4 appears not to affect CTL-mediated immunity. This report investigates these disparate roles of IL-4 in CD8+ T lymphocyte regulation by comparing T cell responses specific for a single HIV-IIIIB gp120-derived epitope in BALB/c mice deficient in IL-4 to those in wild-type controls. CTL activation was monitored during the acute and memory phases following immunization with recombinant vaccinia virus. Similar frequencies of gp120-specific CTL precursors in splenocytes from both groups indicated that IL-4 plays no significant role in either CTL priming or the establishment of memory. However, cytolytic activity in cultures derived from IL-4-deficient mice developed earlier and was strikingly enhanced following in vitro restimulation, an effect exhibited by both primary and memory T cells. Secretion of IL-2 and IFN-γ by CD8+ T cells from IL-4-deficient mice was also elevated, reflecting their enhanced activation. Thus, IL-4 appears to limit the activation, expansion, and differentiation of CD8+ T cells with high cytolytic potential.
Oligoclonality of Rat Intestinal Intraepithelial T Lymphocytes: Overlapping TCR β-Chain Repertoires in the CD4 Single-Positive and CD4/CD8 Double-Positive Subsets
Authors: Helgeland Lars , Johansen Finn-Eirik , Utgaard Jon O. , Vaage John T. , Brandtzaeg Per
Published: December 2022

Previous studies in humans and mice have shown that gut intraepithelial lymphocytes (IELs) express oligoclonal TCR β-chain repertoires. These studies have either employed unseparated IEL preparations or focused on the CD8+ subsets. Here, we have analyzed the TCR β-chain repertoire of small intestinal IELs in PVG rats, in sorted CD4+ as well as CD8+ subpopulations, and important differences were noted. CD8αα and CD8αβ single-positive (SP) IELs used most Vβ genes, but relative Vβ usage as determined by quantitative PCR analysis differed markedly between the two subsets and among individual rats. By contrast, CD4+ IELs showed consistent skewing toward Vβ17 and Vβ19; these two genes accounted collectively for more than half the Vβ repertoire in the CD4/CD8 double-positive (DP) subset and were likewise predominant in CD4 SP IELs. Complementarity-determining region 3 length displays and TCR sequencing demonstrated oligoclonal expansions in both the CD4+ and CD8+ IEL subpopulations. These studies also revealed that the CD4 SP and CD4/CD8 DP IEL subsets expressed overlapping β-chain repertoires. In conclusion, our results show that rat TCR-αβ+ IELs of both the CD8+ and CD4+ subpopulations are oligoclonal. The limited Vβ usage and overlapping TCR repertoire expressed by CD4 SP and CD4/CD8 DP cells suggest that these two IEL populations recognize restricted intestinal ligands and are developmentally and functionally related.
Expression of Dominant-Negative Src-Homology Domain 2-Containing Protein Tyrosine Phosphatase-1 Results in Increased Syk Tyrosine Kinase Activity and B Cell Activation
Authors: Dustin Lynn B. , Plas David R. , Wong Jane , Hu Yonghong Tammy , Soto Carlos , Chan Andrew C. , Thomas Matthew L.
Published: December 2022

The Src-homology domain 2 (SH2)-containing cytoplasmic tyrosine phosphatase, SHP-1 (SH2-containing protein tyrosine phosphatase-1), interacts with several B cell surface and intracellular signal transduction molecules through its SH2 domains. Mice with the motheaten and viable motheaten mutations are deficient in SHP-1 and lack most mature B cells. To define the role of SHP-1 in mature B cells, we expressed phosphatase-inactive SHP-1 (C453S) in a mature B cell lymphoma line. SHP-1 (C453S) retains the ability to bind to both substrates and appropriate tyrosine-phosphorylated proteins and therefore can compete with the endogenous wild-type enzyme. We found that B cells expressing SHP-1 (C453S) demonstrated enhanced and prolonged tyrosine phosphorylation of proteins with molecular masses of 110, 70, and 55–60 kDa after stimulation with anti-mouse IgG. The tyrosine kinase Syk was hyperphosphorylated and hyperactive in B cells expressing SHP-1 (C453S). SHP-1 and Syk were coimmunoprecipitated from wild-type K46 cells, K46 SHP-1 (C453S) cells, and splenic B cells, and SHP-1 dephosphorylated Syk. Cells expressing SHP-1 (C453S) showed increased Ca2+ mobilization, extracellular signal-regulated kinase activation, and homotypic adhesion after B cell Ag receptor engagement. Thus, SHP-1 regulates multiple early and late events in B lymphocyte activation.
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

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

Costimulation (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

The 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

Adhesiveness 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

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

Theradigm-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

Genomic 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

The 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

The 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

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

We 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

Decreased 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

Kaposi’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 >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

The 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.
Induction of Autoimmunity in a Transgenic Model of B Cell Receptor Peripheral Tolerance: Changes in Coreceptors and B Cell Receptor-Induced Tyrosine-Phosphoproteins
Authors: Feuerstein Nili , Chen Fangqi , Madaio Michael , Maldonado Michael , Eisenberg Robert A.
Published: December 2022

Abrogation of peripheral tolerance in transgenic mice that express a uniform B-cell receptor may create a powerful tool to examine the molecular mechanisms that underlie the autoimmune response in B cells. Here we report that processes that induce a systemic lupus erythematosus-like syndrome in normal mice, namely chronic graft vs host reaction, trigger systemic autoimmunity in a well-established transgenic mice model of B cell receptor peripheral tolerance. The induction of graft vs host reaction in mice that carry both a rearranged B cell Ag receptors specific for hen egg lysozyme and expressing chronically circulating hen egg lysozyme Ag resulted in induction of high and sustained levels of circulating anti-hen egg lysoyme autoantibodies and glomerulonephritis with proteinuria. This was associated with marked changes in expression of cell-surface proteins, such as CD23 and complement receptor 2. B cells from the graft vs host-induced mice could proliferate in vitro in response to self-Ag, and upon stimulation with anti-IgD demonstrated rapid phosphotyrosine phosphorylation of specific proteins, which could not be induced in the anergic double transgenic B cells. Conversely, loss of tolerance was not associated with a higher induction in the level of Syk kinase phosphorylation following stimulation with anti-IgD. Taken collectively, these data establish that 1) processes that induce a systemic lupus erythematosus-like syndrome in normal mice can abrogate peripheral tolerance in transgenic mice expressing self-tolerized B cells, and that 2) loss of tolerance in this model is associated with marked changes in surface expression of B cell coreceptors as well as with selective changes in IgD-induced signaling by discrete tyrosine-phosphoproteins, but not Syk kinase.
NK Cell-Deficient Mice Develop a Th1-Like Response but Fail to Mount an Efficient Antigen-Specific IgG2a Antibody Response
Authors: Satoskar Abhay R. , Stamm Luisa M. , Zhang Ximing , Okano Mitsuhiro , David John R. , Terhorst Cox , Wang Baoping
Published: December 2022

NK cells have been shown to play a role in the modulation of B cell differentiation and Ab production. Using a novel murine model of NK cell deficiency, we analyzed the in vivo role of NK cells in the regulation of Ag-specific Ab production. After immunization with OVA or keyhole limpet hemocyanin in CFA, NK cell-deficient (NK−T+) mice developed an efficient Th1 response and produced significant levels of IFN-γ but displayed markedly reduced or absent Ag-specific IgG2a production. There were no differences in the levels of Ag-specific IgG, IgG1, and IgG2b between NK−T+ and NK+T+ mice. Furthermore, NK cell-reconstituted, NK+T+ (tgε26Y) mice produced significant amounts of Ag-specific IgG2a after immunization with OVA. These results indicate that NK cells are involved in the induction of Ag-specific IgG2a production in vivo. Moreover, they also demonstrate that the lack of Ag-specific IgG2a Ab production in NK−T+ mice is not associated with the impaired Th1 response and IFN-γ production.
The Role of CD8α′ in the CD4 Versus CD8 Lineage Choice
Authors: Salmon Patrick , Mong Mimi , Kang Xiao-Jun , Cado Dragana , Robey Ellen
Published: December 2022

During thymic development the recognition of MHC proteins by developing thymocytes influences their lineage commitment, such that recognition of class I MHC leads to CD8 T cell development, whereas recognition of class II MHC leads to CD4 T cell development. The coreceptors CD8 and CD4 may contribute to these different outcomes through interactions with class I and class II MHC, respectively, and through interactions with the tyrosine kinase p56lck (Lck) via their cytoplasmic domains. In this paper we provide evidence that an alternatively spliced form of CD8 that cannot interact with Lck (CD8α′) can influence the CD4 vs CD8 lineage decision. Constitutive expression of a CD8 minigene transgene that encodes both CD8α and CD8α′ restores CD8 T cell development in CD8α mutant mice, but fails to permit the development of mismatched CD4 T cells bearing class I-specific TCRs. These results indicate that CD8α′ favors the development of CD8-lineage T cells, perhaps by reducing Lck activity upon class I MHC recognition in the thymus.
Heterogeneity of NK1.1+ T Cells in the Bone Marrow: Divergence from the Thymus
Authors: Zeng Defu , Gazit Gadi , Dejbakhsh-Jones Sussan , Balk Steven P. , Snapper Scott , Taniguchi Masaru , Strober Samuel
Published: December 2022

NK1.1+ T cells in the mouse thymus and bone marrow were compared because some marrow NK1.1+ T cells have been reported to be extrathymically derived. Almost all NK1.1+ T cells in the thymus were depleted in the CD1−/−, β2m−/−, and Jα281−/− mice as compared with wild-type mice. CD8+NK1.1+ T cells were not clearly detected, even in the wild-type mice. In bone marrow from the wild-type mice, CD8+NK1.1+ T cells were easily detected, about twice as numerous as CD4+NK1.1+ T cells, and were similar in number to CD4−CD8−NK1.1+ T cells. All three marrow NK1.1+ T cell subsets were reduced about 4-fold in CD1−/− mice. No reduction was observed in CD8+NK1.1+ T cells in the bone marrow of Jα281−/− mice, but marrow CD8+NK1.1+ T cells were markedly depleted in β2m−/− mice. All NK1.1+ T cell subsets in the marrow of wild-type mice produced high levels of IFN-γ, IL-4, and IL-10. Although the numbers of marrow CD4−CD8−NK1.1+ T cells in β2m−/− and Jα281−/− mice were similar to those in wild-type mice, these cells had a Th1-like pattern (high IFN-γ, and low IL-4 and IL-10). In conclusion, the large majority of NK1.1+ T cells in the bone marrow are CD1 dependent. Marrow NK1.1+ T cells include CD8+, Vα14-Jα281−, and β2m-independent subsets that are not clearly detected in the thymus.
Generation of Polymeric Immunoglobulin Receptor-Deficient Mouse with Marked Reduction of Secretory IgA
Authors: Shimada Shin-ichiro , Kawaguchi-Miyashita Mariko , Kushiro Akira , Sato Takashi , Nanno Masanobu , Sako Tomoyuki , Matsuoka Yoshiaki , Sudo Katsuko , Tagawa Yoh-ichi , Iwakura Yoichiro , Ohwaki Makoto
Published: December 2022

We generated mouse lacking exon 2 of polymeric Ig receptor (pIgR) gene by a gene-targeting strategy (pIgR-deficient mouse; pIgR−/− mouse) to define the physiological role of pIgR in the transcytosis of Igs. pIgR−/− mice were born at the expected ratio from a cross between pIgR+/− mice, indicating that disruption of the pIgR gene in mice is not lethal. pIgR and secretory component proteins were not detected in pIgR−/− mice by Western blot analysis. Moreover, immunohistochemical analysis showed that pIgR protein is not expressed in jejunal and colonic epithelial cells of pIgR−/− mice, whereas IgA+ cells are present in the intestinal mucosa of pIgR−/− mice as well as wild-type littermates. Disruption of the pIgR gene caused a remarkable increase in serum IgA concentration and a slight increment of serum IgG and IgE levels, leaving serum IgM level unaltered. In contrast, IgA was much reduced but not negligible in the bile, feces, and intestinal contents of pIgR−/− mice. Additionally, IgA with a molecular mass of 280 kDa preferentially accumulated in the serum of pIgR−/− mice, suggesting that transepithelial transport of dIgA is severely blocked in pIgR−/− mice. These results demonstrate that dIgA is mainly transported by pIgR on the epithelial cells of intestine and hepatocytes, but a small quantity of IgA may be secreted via other pathways.
γ-Chain Dependent Recruitment of Tyrosine Kinases to Membrane Rafts by the Human IgA Receptor FcαR
Authors: Lang Mark L. , Shen Li , Wade William F.
Published: December 2022

We show that the human IgA receptor, FcαR, redistributes to plasma membrane rafts after cross-linking and that tyrosine kinases are relocated to these sites following FcαR capping. We demonstrate by confocal microscopy that FcαR caps in membrane rafts by a γ-chain-independent mechanism but that γ-chain expression is necessary for Lyn redistribution. Immunoblotting of rafts isolated by sucrose density gradient centrifugation demonstrated recruitment of γ-chain and phosphorylated tyrosine kinases Lyn and Bruton’s tyrosine kinase to membrane rafts after FcαR cross-linking. Time-dependent differences in Lyn phosphorylation and Bruton’s tyrosine kinase distribution were observed between cells expressing FcαR plus γ-chain and cells expressing FcαR only. This study defines early FcαR-triggered membrane dynamics that take place before FcαR internalization.
Reactive Oxygen Intermediates During Programmed Cell Death Induced in the Thymus of the Ts(1716)65Dn Mouse, a Murine Model for Human Down’s Syndrome
Authors: Paz-Miguel Jesús E. , Flores Reyes , Sánchez-Velasco Pablo , Ocejo-Vinyals Gonzalo , Escribano de Diego Juan , López de Rego Jacobo , Leyva-Cobián Francisco
Published: December 2022

Down’s syndrome (DS) is one of the most frequent genetic disorders in humans. It has been suggested that overexpression of copper-zinc superoxide dismutase (SOD-1) in DS may be involved in some of the abnormalities observed, mainly neurodegenerative and immunopathological processes. One of the consequences is early thymic involution. Recently, Ts(1716)65Dn mice (Ts65Dn mice), made segmentally trisomic for a chromosome 16 segment, fulfill the criteria for a DS model. To study the possible role of SOD-1 overexpression in thymocyte biology, we analyzed the role of reactive oxygen intermediates during in vivo and in vitro programmed cell death (PCD) induced in the thymus of Ts65Dn mice. Our main findings can be summarized as follows. Ts65Dn thymuses exhibit greater PCD activity than controls, as ascertained by a combination of morphological, histochemical, and ultrastructural procedures. Ts65Dn thymocytes were highly susceptible to PCD induced by both LPS (in vivo) and dexamethasone, a synthetic glucocorticoid agonist (both in vivo and in vitro). Thymus abnormalities were probably caused by SOD-1 hyperexpression in Ts65Dn cells, in that reactive oxygen intermediate generation (specifically H2O2 production) is enhanced in thymocytes and clearly correlates with apoptosis. Similarly, oxidative injury correlated with the formation of lipid peroxidation by-products and antioxidants which partly inhibit PCD in thymocytes.
Differential Activation of Mitogen-Activated Protein Kinases in AGS Gastric Epithelial Cells bycag+ andcag− Helicobacter pylori
Authors: Keates Sarah , Keates Andrew C. , Warny Michel , Peek Richard M. , Murray Paul G. , Kelly Ciarán P.
Published: December 2022

The aim of this study was to determine whether Helicobacter pylori activates mitogen-activated protein (MAP) kinases in gastric epithelial cells. Infection of AGS cells with an H. pylori cag+ strain rapidly (5 min) induced a dose-dependent activation of extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK) MAP kinases, as determined by Western blot analysis and in vitro kinase assay. Compared with cag+ strains, cag− clinical isolates were less potent in inducing MAP kinase, particularly JNK and p38, activation. Isogenic inactivation of the picB region of the cag pathogenicity island resulted in a similar loss of JNK and p38 MAP kinase activation. The specific MAP kinase inhibitors, PD98059 (25 μM; MAP kinase kinase (MEK-1) inhibitor) and SB203580 (10 μM; p38 inhibitor), reduced H. pylori-induced IL-8 production in AGS cells by 78 and 82%, respectively (p < 0.01 for each). Both inhibitors together completely blocked IL-8 production (p < 0.001). However, the MAP kinase inhibitors did not prevent H. pylori-induced IκBα degradation or NF-κB activation. Thus, H. pylori rapidly activates ERK, p38, and JNK MAP kinases in gastric epithelial cells; cag+ isolates are more potent than cag− strains in inducing MAP kinase phosphorylation and gene products of the cag pathogenicity island are required for maximal MAP kinase activation. p38 and MEK-1 activity are required for H. pylori-induced IL-8 production, but do not appear to be essential for H. pylori-induced NF-κB activation. Since MAP kinases regulate cell proliferation, differentiation, programmed death, stress, and inflammatory responses, activation of gastric epithelial cell MAP kinases by H. pylori cag+ strains may be instrumental in inducing gastroduodenal inflammation, ulceration, and neoplasia.
Modulation of Chemokine Expression During Ischemia/Reperfusion in Transgenic Mice Overproducing Human Glutathione Peroxidases
Authors: Ishibashi Nobuya , Weisbrot-Lefkowitz Miriam , Reuhl Kenneth , Inouye Masayori , Mirochnitchenko Oleg
Published: December 2022

Renal ischemia/reperfusion (I/R) injury is a major cause of kidney damage. There is accumulating evidence that inflammatory reactions are involved in the pathogenesis of this process. Our studies demonstrate that transgenic mice overexpressing human extracellular and intracellular glutathione peroxidases (GP) are protected against kidney I/R injury. Importantly, significant reduction in neutrophil migration was observed in GP mice compared with nontransgenic mice. Analysis of signaling molecules mediating neutrophil activation and recruitment indicates reduction in the level of KC and macrophage inflammatory protein-2 chemokine expression in transgenic animals. The molecular mechanism mediating this effect appears to involve repression of NF-κB activation at the level of IκBα and IκBβ degradation. In the case of IκBα, no apparent phosphorylation was detected. These results suggest that IκBα proteolysis is triggered during the renal I/R pro-oxidant state by a still unknown mechanism, which might be different from other stimuli. A central role of NF-κB in CXC chemokine activation was demonstrated in cell culture anoxia/ATP repletion experiments as a model of I/R. The data presented indicate the important role of GP-sensitive signal transduction pathways in the development of inflammatory response and tissue injury during I/R.
Isolation of Self Antigen-Reactive Cells from Inflamed Islets of Nonobese Diabetic Mice Using CD4high Expression as a Marker
Authors: Lejon C. Kristina , Fathman Garrison
Published: December 2022

The low precursor frequency of Ag-reactive CD4+ T cells has been a barrier to the study of CD4+ T cell responses to conventional Ags as well as CD4+ T cell responses to autoantigens recognized during the course of an autoimmune disease. We have recently reported that all “conventional Ag” reactive CD4+ T cells are contained within the subpopulation expressing high levels of the CD4 molecule, termed CD4high. We have identified a CD4high population in the islets of Langerhans of prediabetic nonobese diabetic (NOD) mice that is extremely potent in transferring disease. As few as 500 CD4high islet-infiltrating CD4+ T cells transferred insulin-dependent diabetes mellitus to CD8 reconstituted NOD-SCID mice within 30 days of transfer. In contrast, CD4high T cells isolated from either NOD spleen or salivary glands did not transfer insulin-dependent diabetes mellitus into similar CD8-reconstituted NOD-SCID recipients. These data indicate that the precursor frequency of NOD islet-reactive, pathogenic CD4+ T cells is much higher in the prediabetic NOD pancreas than in these other organs. The islet-infiltrating CD4high T cells displayed selected memory markers, by cell surface analysis, and displayed a Th 1 phenotype by RNase protection assay, but had a marked decrease in IL-4 mRNA determined by quantitative real time PCR when compared with the less pathogenic CD4normal islet-infiltrating T cells. Use of the CD4high marker to select Ag activated T cells represents a tool to isolate and study pathogenic CD4+ T cells from autoimmune lesions in which the Ag has not been previously defined.
Transfer of the Enhancing Effect of Respiratory Syncytial Virus Infection on Subsequent Allergic Airway Sensitization by T Lymphocytes
Authors: Schwarze Jürgen , Makela Mika , Cieslewicz Grzegorz , Dakhama Azzeddine , Lahn Michael , Ikemura Toshihide , Joetham Anthony , Gelfand Erwin W.
Published: December 2022

In mice, respiratory syncytial virus (RSV) infection enhances allergic airway sensitization, resulting in lung eosinophilia and in airway hyperresponsiveness (AHR). The mechanisms by which RSV contributes to development of asthma and its effects on allergic airway sensitization in mice are not known. We tested whether these consequences of RSV infection can be adoptively transferred by T cells and whether depletion of T cell subsets prevents the effects of RSV infection on subsequent airway sensitization. Mononuclear cells, T lymphocytes, or CD4 or CD8 T cells from peribronchial lymph nodes (PBLN) of RSV-infected mice were transferred into naive BALB/c mice which were then exposed to OVA via the airways. Additionally, RSV-infected mice were depleted of CD4 or CD8 T cells following acute RSV infection but prior to airway sensitization. Following sensitization, airway responsiveness to inhaled methacholine, numbers of lung eosinophils, and levels of IFN-γ, IL-4, and IL-5 in PBLN cell cultures were monitored. Transfer of T cells from RSV-infected mice resulted in increased eosinophil influx into the lungs, increased IL-5 production, and development of AHR following airway sensitization to allergen. Transfer of CD8 but not CD4 T cells from the PBLN of RSV-infected mice also resulted in AHR following 10 days of OVA exposure. Further, depletion of CD8 T cells prevented these consequences of RSV infection while CD4 T cell depletion reduced them. We conclude that T cells, in particular CD8 T cells, are critical in mediating RSV-induced development of lung eosinophilia and AHR following allergic airway sensitization.
Antigen-Driven Expansion and Contraction of CD8+-Activated T Cells in Primary EBV Infection
Authors: Hoshino Yo , Morishima Tsuneo , Kimura Hiroshi , Nishikawa Kazuo , Tsurumi Tatsuya , Kuzushima Kiyotaka
Published: December 2022

The origin of the increased numbers of CD8+ atypical lymphocytes, expressing activated markers such as HLA-DR or CD45RO, in the peripheral blood of patients with infectious mononucleosis (IM) has been debated. Using a recently developed assay to detect intracellular accumulation of IFN-γ in EBV-reactive T cells by FACS, we have demonstrated that 34–54% of HLA-DR+/CD8+ and 34–60% of CD45RO+/CD8+ T cells in the PBMCs of febrile patients suffering from IM are EBV-specific. The EBV-specific CD8+ T cell counts in the PBMCs of four febrile patients suffering from IM ranged between 2260 and 8200/μl, decreasing to 5.1% and 7.9% of the counts in the first samples over 10 days in two donors. The decline of CD8+ T cell subpopulations, namely HLA-DR+, CD45RO+, and EBV-specific T cells, was in parallel with the drop in the EBV genome load. These data indicate that the Ag-driven expansion of CD8+ T cells and subsequent contraction with the Ag decline in vivo in humans is effective for clearing virus-infected cells with minimal disturbance of the homeostasis of the immune system.