bluuebunny/crossref_metadata_2025_split huggingface dataset:
CD45 Is Essential for FcεRI Signaling by ZAP70, But Not Syk, in Syk-Negative Mast Cells
Authors: Zhang Juan , Siraganian Reuben P. 
Published: December 2022

AbstractThe ZAP70/Syk family of protein tyrosine kinases plays an important role in Ag receptor signaling. Structural similarity of Syk and ZAP70 suggests their functional overlap. Previously, it was observed that expression of either ZAP70 or Syk reconstitutes Ag receptor signaling in Syk-negative B cells. However, in CD45-deficient T cells, Syk, but not ZAP70, restores T cell receptor-signaling pathway. To study the function of Syk, ZAP70, and CD45 in mast cells, a Syk/CD45 double-deficient variant of RBL-2H3 cells was characterized. After transfection, stable cell lines were isolated that expressed ZAP70, Syk, CD45, ZAP70 plus CD45, and Syk plus CD45. IgE stimulation did not induce degranulation in parental double-deficient cells, nor in the cells expressing only CD45. ZAP70 expression did not restore FcεRI signaling unless CD45 was coexpressed in the cells. However, Syk alone restored the IgE signal transduction pathway. The coexpression of CD45 with Syk had no significant effects on the responses to FcεRI-aggregation. There was much better binding of Syk than ZAP70 to the phosphorylated FcεRIγ-ITAM. Furthermore, unlike Syk, ZAP70 required CD45 to display receptor-induced increase in kinase activity. Therefore, in mast cells, ZAP70, but not Syk, requires CD45 for Ag receptor-induced signaling.
Phenotypical and Functional Characterization of the CD8+ T Cell Repertoire ofHLA-A2.1Transgenic,H-2Kb°Db°Double Knockout Mice
Authors: Ureta-Vidal Abel , Firat Hüseyin , Pérarnau Béatrice , Lemonnier François A. 
Published: December 2022

AbstractHomozygous HLA-A2.1 transgenic H-2Kb°Db° double knockout (KO) mice were created. Their potential to develop HLA-A2.1-restricted cytolytic responses was compared with that of their classical transgenic counterparts, which still express H-2Kb, Db molecules. On cell surfaces, both strains express similar amounts of chimeric (α1α2 domains of human, α3 cytoplasmic domains of mouse) HLA-A2.1 molecules in noncovalent association with mouse β2-microglobulin. Compared with mice that are totally deprived of histocompatibility class Ia molecules (H-2Kb°Db° double KO), the expression of HLA-A2.1 in transgenic/double KO mice resulted in sizeable increase in the periphery of CD8+ T cells with a normally diversified TCR repertoire. A biased education in favor of HLA-A2.1, ascribable to the absence of H-2 class Ia molecules, was evidenced in these transgenic/double KO mice by their improved capacity to mount HLA-restricted cytolytic responses, regardless of whether they were virally infected or injected with synthetic epitopic peptide. HLA class I transgenic, H-2 class Ia KO mice should represent useful animal models for the preclinical evaluation of vaccine formulations aiming at the induction of HLA class I-restricted CTL responses.
A Role for the Transmembrane Domain in the Trimerization of the MHC Class II-Associated Invariant Chain
Authors: Ashman Jonathan B. , Miller Jim 
Published: December 2022

AbstractMHC class II and invariant chain (Ii) associate early in biosynthesis to form a nonameric complex. Ii first assembles into a trimer and then associates with three class II αβ heterodimers. Although the membrane-proximal region of the Ii luminal domain is structurally disordered, the C-terminal segment of the luminal domain is largely α-helical and contains a major interaction site for the Ii trimer. In this study, we show that the Ii transmembrane domain plays an important role in the formation of Ii trimers. The Ii transmembrane domain contains an unusual patch of hydrophilic residues near the luminal interface. Substitution of these polar residues with nonpolar amino acids resulted in a decrease in the efficiency of Ii trimerization and subsequent class II association. Moreover, N-terminal fragments of Ii were found to trimerize independently of the luminal α-helical domain. Progressive C-terminal truncations mapped a homotypic association site to the first 80 aa of Ii. Together, these results implicate the Ii transmembrane domain as a site of trimer interaction that can play an important role in the initiation of trimer formation.
Differential Expression and Distinct Functions of IFN Regulatory Factor 4 and IFN Consensus Sequence Binding Protein in Macrophages
Authors: Marecki Sylvia , Atchison Michael L. , Fenton Matthew J. 
Published: December 2022

AbstractIFN regulatory factor 4 (IRF4) and IFN consensus sequence binding protein (ICSBP) are highly homologous members of the growing family of IRF proteins. ICSBP expression is restricted to lymphoid and myeloid cells, whereas IRF4 expression has been reported to be lymphoid-restricted. We present evidence that primary murine and human macrophages express IRF4, thereby extending its range of expression to myeloid cells. Here, we provide a comparative analysis of IRF4 and ICSBP expression and function in distinct cell types. These IRF proteins can form specific complexes with the Ets-like protein PU.1, and can activate transcription via binding to PU.1/IRF composite sequences. EMSA analysis revealed that murine macrophages contained both IRF4/PU.1 and ICSBP/PU.1 complexes, analogous to B cells. Over-expression of ICSBP in these macrophages activated transcription of a PU.1/IRF-dependent promoter, whereas over-expression of IRF4 had no effect on this promoter. In contrast, over-expression of either IRF4 or ICSBP in both macrophages and NIH-3T3 fibroblasts suppressed transcription of the PU.1-independent H-2Ld MHC class I promoter. In NIH-3T3 fibroblasts, IRF4 and ICSBP also synergized with exogenous PU.1 to activate transcription of a PU.1/IRF-dependent promoter. Furthermore, both IRF4 and ICSBP activated transcription of the IL-1β promoter in both cell types. While this promoter is PU.1-dependent, it lacks any known PU.1/IRF composite binding sites. Synergistic activation of the IL-1β promoter by these IRF proteins and PU.1 was found to require PU.1 serine 148. Together, these data demonstrate that IRF4 and ICSBP are dichotomous regulators of transcription in macrophages.
Role of IL-10 in a Neonatal Mouse Listeriosis Model
Authors: Genovese Francesco , Mancuso Giuseppe , Cuzzola Maria , Biondo Carmelo , Beninati Concetta , Delfino Demetrio , Teti Giuseppe 
Published: December 2022

AbstractThis study was undertaken to test the hypothesis that altered IL-10 production plays a role in the increased susceptibility of neonates to listeriosis. Plasma IL-10 levels were measured in neonatal and adult mice at various times after infection with Listeria monocytogenes. Relative to adults, neonatal mice had markedly increased IL-10 levels early in the course of infection with Listeria using a 90% lethal dose. Higher neonatal IL-10 responses were also observed after injecting adults and pups with equal doses of killed organisms. Splenic macrophages from neonates produced higher IL-10 levels than those of adults after in vitro stimulation with killed bacteria, confirming in vivo observations. Moreover, IL-10 blockade had differential effects in neonates and adults infected with live Listeria. In adult mice, anti-IL-10 Abs decreased bacterial burden early in the course of infection, but were no longer effective at 6 days or later after challenge. In the pups, however, the same treatment had beneficial effects both early and late during infection and resulted in increased survival. Collectively, our data suggest that an overproduction of IL-10 by macrophages may at least partially explain the increased susceptibility of neonates to listeriosis, and provide further evidence that cytokine production is different in adults and neonates.
Human Dendritic Cells Very Efficiently Present a Heterologous Antigen Expressed on the Surface of Recombinant Gram-Positive Bacteria to CD4+ T Lymphocytes
Authors: Corinti Silvia , Medaglini Donata , Cavani Andrea , Rescigno Maria , Pozzi Gianni , Ricciardi-Castagnoli Paola , Girolomoni Giampiero 
Published: December 2022

AbstractRecombinant Streptococcus gordonii expressing on the surface the C-fragment of tetanus toxin was tested as an Ag delivery system for human monocyte-derived dendritic cells (DCs). DCs incubated with recombinant S. gordonii were much more efficient than DCs pulsed with soluble C-fragment of tetanus toxin at stimulating specific CD4+ T cells as determined by cell proliferation and IFN-γ release. Compared with DCs treated with soluble Ag, DCs fed with recombinant bacteria required 102- to 103-fold less Ag and were at least 102 times more effective on a per-cell basis for activating specific T cells. S. gordonii was internalized in DCs by conventional phagocytosis, and cytochalasin D inhibited presentation of bacteria-associated Ag, but not of soluble Ag, suggesting that phagocytosis was required for proper delivery of recombinant Ag. Bacteria were also very potent inducers of DC maturation, although they enhanced the capacity of DCs to activate specific CD4+ T cells at concentrations that did not stimulate DC maturation. In particular, S. gordonii dose-dependently up-regulated expression of membrane molecules (MHC I and II, CD80, CD86, CD54, CD40, CD83) and reduced both phagocytic and endocytic activities. Furthermore, bacteria promoted in a dose-dependent manner DC release of cytokines (IL-6, TNF-α, IL-1β, IL-12, TGF-β, and IL-10) and of the chemokines IL-8, RANTES, IFN-γ-inducible protein-10, and monokine induced by IFN-γ. Thus, recombinant Gram-positive bacteria appear a powerful tool for vaccine design due to their extremely high capacity to deliver Ags into DCs, as well as induce DC maturation and secretion of T cell chemoattractans.
Migration of Antigen-Presenting B Cells from Peripheral to Mucosal Lymphoid Tissues May Induce Intestinal Antigen-Specific IgA Following Parenteral Immunization
Authors: Coffin Susan E. , Clark Stephanie L. , Bos Nico A. , Brubaker Jeffery O. , Offit Paul A. 
Published: December 2022

AbstractParenterally administered immunizations have long been used to induce protection from mucosal pathogens such as Bordetella pertussis and influenza virus. We previously found that i.m. inoculation of mice with the intestinal pathogen, rotavirus, induced virus-specific Ab production by intestinal lymphocytes. We have now used adoptive transfer studies to identify the cell types responsible for the generation of virus-specific Ab production by gut-associated lymphoid tissue (GALT) after i.m. immunization. Three days after i.m. immunization with rotavirus, cells obtained from the draining peripheral lymph nodes of donor mice were transferred into naive recipient mice. We found that intestinal lymphocytes produced rotavirus-specific Igs (IgM, IgA, and IgG) 2 wk after transfer of either unfractionated cells, or unfractionated cells rendered incapable of cellular division by mitomycin C treatment. Additional studies demonstrated that rotavirus-specific IgA, but not IgG, was produced by intestinal lymphocytes after transfer of purified B cells. Ig allotype analysis revealed that rotavirus-specific IgA was produced by intestinal B cells of recipient origin, suggesting that migration of Ag-presenting B cells from peripheral lymphoid tissues to GALT may contribute to the generation of mucosal IgA responses after parenteral immunization. Strategies that promote Ag uptake and presentation by B cells may enhance mucosal IgA production following parenteral immunization.
IL-4-Producing γδ T Cells That Express a Very Restricted TCR Repertoire Are Preferentially Localized in Liver and Spleen
Authors: Gerber David J. , Azuara Véronique , Levraud Jean-Pierre , Huang Shu Ying , Lembezat Marie-Pierre , Pereira Pablo 
Published: December 2022

AbstractIL-4-producing γδ thymocytes in normal mice belong to a distinct subset of γδ T cells characterized by low expression of Thy-1. This γδ thymocyte subset shares a number of phenotypic and functional properties with the NK T cell population. Thy-1dull γδ thymocytes in DBA/2 mice express a restricted repertoire of TCRs that are composed of the Vγ1 gene product mainly associated with the Vδ6.4 chain and exhibit limited junctional sequence diversity. Using mice transgenic for a rearranged Vγ1Jγ4Cγ4 chain and a novel mAb (9D3) specific for the Vδ6.3 and Vδ6.4 murine TCRδ chains, we have analyzed the peripheral localization and functional properties of γδ T cells displaying a similarly restricted TCR repertoire. In transgenic mice, IL-4 production by peripheral γδ T cells was confined to the γδ+9D3+ subset, which contains cells with a TCR repertoire similar to that found in Thy-1dull γδ thymocytes. In normal DBA/2 mice such cells represent close to half of the γδ T cells present in the liver and around 20% of the splenic γδ T cells.
The Role of the Common Cytokine Receptor γ-Chain in Regulating IL-2-Dependent, Activation-Induced CD8+ T Cell Death
Authors: Dai Zhenhua , Arakelov Alexandr , Wagener Maylene , Konieczny Bogumila T. , Lakkis Fadi G. 
Published: December 2022

AbstractIL-2-dependent, activation-induced T cell death (AICD) plays an important role in peripheral tolerance. Using CD8+ TCR-transgenic lymphocytes (2C), we investigated the mechanisms by which IL-2 prepares CD8+ T cells for AICD. We found that both Fas and TNFR death pathways mediate the AICD of 2C cells. Neutralizing IL-2, IL-2Rα, or IL-2Rβ inhibited AICD. In contrast, blocking the common cytokine receptor γ-chain (γc) prevented Bcl-2 induction and augmented AICD. IL-2 up-regulated Fas ligand (FasL) and down-regulated γc expression on activated 2C cells in vitro and in vivo. Adult IL-2 gene-knockout mice displayed exaggerated γc expression on their CD8+, but not on their CD4+, T cells. IL-4, IL-7, and IL-15, which do not promote AICD, did not influence FasL or γc expression. These data provide evidence that IL-2 prepares CD8+ T lymphocytes for AICD by at least two mechanisms: 1) by up-regulating a pro-apoptotic molecule, FasL, and 2) by down-regulating a survival molecule, γc.
SLP-76 Binding to p56lck: A Role for SLP-76 in CD4-Induced Desensitization of the TCR/CD3 Signaling Complex
Authors: Sanzenbacher Ralf , Kabelitz Dieter , Janssen Ottmar 
Published: December 2022

AbstractNonreceptor protein tyrosine kinases and associated substrates play a pivotal role in Ag receptor stimulation of resting cells and in the initiation of activation-induced cell death (AICD) of preactivated T cells. CD4-associated p56lck has been implicated not only in the activation of primary T cells, but also in the inhibition of T cell responses. We have previously shown that CD4+ T cell clones can be rescued from AICD when surface CD4 is engaged before the TCR stimulus. In this study, we show that prevention of AICD is associated with a CD4-dependent inhibition of TCR-triggered tyrosine phosphorylation of the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) and Vav. We provide evidence for a SLP-76 interaction with Src homology 3 domains of p56lck and identify amino acids 185–194 of SLP-76 as relevant docking site. In view of the multiple functions of p56lck and SLP-76/Vav in the initiation of TCR/CD3/CD4 signaling, we propose a model for the CD4-dependent inhibition of TCR signaling and AICD of preactivated T cells. Our data suggest that preformed activation complexes of adapter proteins and enzymes in the vicinity of the CD4/p56lck complex are no longer available for the TCR signal when CD4 receptors are engaged before TCR stimulation.
B Cell Antigen Receptor-Mediated Activation of Cyclin-Dependent Retinoblastoma Protein Kinases and Inhibition by Co-Cross-Linking with Fcγ Receptors
Authors: Tanguay Debra , Pavlovic Sandra , Piatelli Michael J. , Bartek Jiri , Chiles Thomas C. 
Published: December 2022

AbstractCross-linking the B cell Ag receptor (BCR) to surface Fc receptors for IgG (FcγR) inhibits G1-to-S progression; the mechanism by which this occurs is not completely known. We investigated the regulation of three key cell cycle regulatory components by BCR-FcγR co-cross-linking: G1-cyclins, cyclin-dependent kinases (Cdks), and the retinoblastoma gene product (Rb). Rb functions to suppress G1-to-S progression in mammalian cells. Rb undergoes cell-cycle-dependent phosphorylation, leading to its inactivation and thereby promoting S phase entry. We demonstrate in this paper for the first time that BCR-induced Rb phosphorylation is abrogated by co-cross-linking with FcγR. The activation of Cdk4/6- and Cdk2-dependent Rb protein kinases is concomitantly blocked. FcγR-mediated inhibition of Cdk2 activity results in part from an apparent failure to express Cdk2 protein. By contrast, inhibition of Cdk4/6 activities is not due to suppression of Cdk4/6 or cyclins D2/D3 expression or inhibition of Cdk-activating kinase activity. Cdk4- and Cdk6-immune complexes recovered from B cells following BCR-FcγR co-cross-linking are devoid of coprecipitated D-type cyclins, indicating that inhibition of their Rb protein kinase activities is due in part to the absence of bound D-type cyclin. Thus, BCR-derived activation signals that up-regulate D-type cyclin and Cdk4/6 protein expression remain intact; however, FcγR-mediated signals block cyclin D-Cdk4/6 assembly or stabilization. These results suggest that assembly or stabilization of D-type cyclin holoenzyme complexes 1) is an important step in the activation of Cdk4/6 by BCR signals, and 2) suffice in providing a mechanism to account for inhibition of BCR-stimulated Rb protein phosphorylation by FcγR.
Roles of Integrins and CD44 on the Adhesion and Migration of Fetal Liver Cells to the Fetal Thymus
Authors: Kawakami Naoto , Nishizawa Fumiko , Sakane Naoki , Iwao Mutsumi , Tsujikawa Kazutake , Ikawa Masahito , Okabe Masaru , Yamamoto Hiroshi 
Published: December 2022

AbstractAdhesion and migration of mouse fetal liver (FL) cells to the thymus were investigated using cells from green fluorescent protein transgenic (GFP+) mice. FL cells from GFP+ embryos at 12 gestational days (E12) of mice were incubated with 2′-deoxyguanosine-treated fetal thymus lobe (from E14) by thymic repopulation (hanging drop) culture methods. GFP+ cells were observed in the thymus lobe at the end of the repopulation culture period. A large part of the infiltrated cells expressed CD44 until day 2 of culture on a permeable membrane, then lost the expression. CD25 expression was observed from day 1 to day 4. Around day 8, GFP+ cells became both CD4+ and CD8+. The results support the early observation of the sequential expression of CD44, CD25, and CD4/8 during the early stages of thymocyte development. When anti-CD44 mAb was added at the beginning of the repopulation culture period, GFP+ FL cells adhered to the surface of the thymus lobe but did not migrate into the thymus. Pretreatment of the thymus with hyaluronidase or hyaluronate produced results similar to the results of anti-CD44 treatment. On the other hand, the addition of anti-integrin α4 mAb inhibited adhesion to the thymus, and almost no GFP+ cells were seen on the surface of the thymus lobe. The data suggest that integrin α4 and CD44 play different roles, i.e., integrin α4 is required for the adhesion of FL cells to the thymus lobe and CD44 is required for the migration of the cells into the thymus.
A Novel Role for HEB Downstream or Parallel to the Pre-TCR Signaling Pathway During αβ Thymopoiesis
Authors: Barndt Robert , Dai Mei-Fang , Zhuang Yuan 
Published: December 2022

Abstract
TCR gene rearrangement and expression are central to the development of clonal T lymphocytes. The pre-TCR complex provides the first signal instructing differentiation and proliferation events during the transition from CD4−CD8−TCR− double negative (DN) stage to CD4+CD8+ double positive (DP) stage. How the pre-TCR signal leads to downstream gene expression is not known. HeLa E-box binding protein (HEB), a basic helix-loop-helix transcription factor, is abundantly detected in thymocytes and is thought to regulate E-box sites present in many T cell-specific gene enhancers, including TCR-α, TCR-β, and CD4. Targeted disruption of HEB results in a 5- to 10-fold reduction in thymic cellularity that can be accounted for by a developmental block at the DN to DP stage transition. Specifically, a dramatic increase in the CD4low/−CD8+CD5lowHSA+TCRlow/− immature single positive population and a concomitant decrease in the subsequent DP population are observed. Adoptive transfer test shows that this defect is cell-autonomous and restricted to the αβ T cell lineage. Introduction of an αβ TCR transgene into the HEBko/ko background is not sufficient to rescue the developmental delay. In vivo CD3 cross-linking analysis of thymocytes indicates that TCR signaling pathway in the HEBko/ko mice appears intact. These findings suggest an essential function of HEB in early T cell development, downstream or parallel to the pre-TCR signaling pathway.
Cytosolic Phospholipase A2 Activation Is Essential for β1 and β2 Integrin-Dependent Adhesion of Human Eosinophils
Authors: Zhu Xiangdong , Muñoz Nilda M. , Kim Kwang Pyo , Sano Hiroyuki , Cho Wonhwa , Leff Alan R. 
Published: December 2022

AbstractWe examined the role of cytosolic phospholipase A2 (cPLA2) during human eosinophil adherence to ICAM-1- or VCAM-1-coated plates. IL-5-stimulated eosinophils adhered to ICAM-1 through the β2 integrin CD11b/CD18, while nonstimulated eosinophils did not. By contrast, nonstimulated eosinophils adhered to VCAM-1 through the β1-integrin VLA-4/CD29. Both IL-5-induced adhesion to ICAM-1 and spontaneous adhesion to VCAM-1 corresponded temporally to cPLA2 phosphorylation, which accompanied enhanced catalytic activity of cPLA2. The structurally unrelated cPLA2 inhibitors, arachidonyl trifluoromethylketone and surfactin, significantly inhibited eosinophil adhesion to ICAM-1 and VCAM-1 in a concentration-dependent manner. Inhibition of secretory PLA2, 5-lipoxygenase, or cyclooxygenase did not affect eosinophil adhesion. Addition of arachidonic acid to eosinophils after cPLA2 inhibition with arachidonyl trifluoromethylketone or surfactin did not reverse the blockade of adhesion to ICAM-1 or VCAM-1. However, CV-6209, a receptor-specific antagonist of platelet-activating factor, inhibited all integrin-mediated adhesion. The activated conformation of CD11b as identified by the mAb, CBRM1/5, as well as quantitative surface CD11b expression were up-regulated after IL-5 stimulation. However, cPLA2 inhibition neither prevented CBRM1/5 expression nor blocked surface Mac-1 up-regulation caused by IL-5. Our data suggest that cPLA2 activation and its catalytic product platelet-activating factor play an essential role in regulating β1 and β2 integrin-dependent adhesion of eosinophils. This blockade occurs even in the presence of up-regulated eosinophil surface integrin.