bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential Expression of the Inhibitory IgG Fc Receptor FcγRIIB on Germinal Center Cells: Implications for Selection of High-Affinity B Cells
Authors: Rao Sambasiva P. , Vora Kalpit A. , Manser Tim 
Published: April 2014

AbstractThe murine low-affinity receptor for IgG, FcγRIIB, mediates inhibition of B cell receptor-triggered events in primary B cells. We investigated the expression of FcγRIIB on germinal center (GC) cells to better understand its role in memory B cell development. Immunohistological analyses demonstrated differential regulation of FcγRIIB on GC cells. Its levels are markedly down-regulated on GC B cells and up-regulated on follicular dendritic cells (FDC) at all times during the GC response. Analyses of surface expression of FcγRIIB by flow cytometry and FcγRIIB mRNA levels by RT-PCR analysis confirmed that this FcR is down-regulated in GC B cells. In mice lacking FcγRIIB, the development of the secondary FDC reticulum in GCs is substantially delayed, although the overall kinetics of the GC response are unaltered. These findings have direct implications for models proposed to account for the selection of high-affinity B cells in the GC and suggest a role for FcγRIIB in promoting the maturation of the FDC reticulum.
Sonic Hedgehog Promotes Cell Cycle Progression in Activated Peripheral CD4+ T Lymphocytes
Authors: Lowrey Jacqueline A. , Stewart Gareth A. , Lindey Susannah , Hoyne Gerard F. , Dallman Margaret J. , Howie Sarah E. M. , Lamb Jonathan R. 
Published: April 2014

Abstract
Sonic hedgehog (Shh) signaling is important in the growth and differentiation of many cell types and recently has been reported to play a role in T cell development in the thymus. This prompted us to investigate whether or not Shh contributes to the clonal expansion of peripheral CD4+ T cells. In this study, we demonstrate that Shh and other components of the signaling pathway patched, smoothened, and Gli1 (glioma-associated oncogene) are expressed in peripheral CD4+ T cells. The addition of the biologically active amino-terminal Shh peptide had no effect on resting CD4+ T cells, but significantly enhanced proliferation of anti-CD3/28 Ab-activated CD4+ T cells. This was not due to antiapoptotic effects, but by promoting entry of T cells into the S-G2 proliferative phase of the cell cycle. Neutralizing anti-Shh Ab reduced T cell proliferation by inhibiting cell transition into the S-G2 phase, suggesting that endogenously produced Shh plays a physiological role in the clonal expansion of T cells. Furthermore, we have observed a significant up-regulation of Shh and Gli1 (glioma-associated oncogene) mRNA in activated CD4+ T cells with or without addition of exogenous Shh, which corresponds with maximal CD4+ T cell proliferation, whereas bcl-2 was only up-regulated in activated cells in the presence of Shh. Our findings suggest that endogenously produced Shh may play a role in sustaining normal CD4+ T cell proliferation and exogenously added Shh enhances this response.
The Human IL-3 Locus Is Regulated Cooperatively by Two NFAT-Dependent Enhancers That Have Distinct Tissue-Specific Activities
Authors: Hawwari Abbas , Burrows Joanna , Vadas Mathew A. , Cockerill Peter N. 
Published: April 2014

AbstractThe human IL-3 gene is expressed by activated T cells, mast cells, and eosinophils. We previously identified an enhancer 14 kb upstream of the IL-3 gene, but this element only functioned in a subset of T cells and not in mast cells. To identify additional mechanisms governing IL-3 gene expression, we mapped DNase I hypersensitive (DH) sites and evolutionarily conserved DNA sequences in the IL-3 locus. The most conserved sequence lies 4.5 kb upstream of the IL-3 gene and it encompassed an inducible cyclosporin A-sensitive DH site. A 245-bp fragment spanning this DH site functioned as a cyclosporin A-sensitive enhancer, and was induced by calcium and kinase signaling pathways in both T cells and mast cells via an array of three NFAT sites. The enhancer also encompassed AML1, AP-1, and Sp1 binding sites that potentially mediate function in both T and myeloid lineage cells, but these sites were not required for in vitro enhancer function in T cells. In stably transfected T cells, the −4.5-kb enhancer cooperated with the −14-kb enhancer to activate the IL-3 promoter. Hence, the IL-3 gene is regulated by two enhancers that have distinct but overlapping tissue specificities. We also identified a prominent constitutive DH site at −4.1 kb in T cells, mast cells, and CD34+ myeloid cells. This element lacked in vitro enhancer function, but may have a developmental role because it appears to be the first DH site to exist upstream of the IL-3 gene during hemopoietic development before IL-3 expression.
T Cell Recognition of an Engineered MHC Class I Molecule: Implications for Peptide-Independent Alloreactivity
Authors: Janković Vladimir , Remus Kristin , Molano Alberto , Nikolich-Žugich Janko 
Published: April 2014

AbstractPreviously, we described H-2KbW9 (KbW9), an engineered variant of the murine MHC class I molecule H-2Kb (Kb), devoid of the central anchor (“C”) pocket owing to a point mutation on the floor of the peptide binding site; this substitution drastically altered selection of bound peptides, such that the peptide repertoires of Kb and KbW9 are largely nonoverlapping in vivo. On the basis of these observations, we used KbW9 and Kb to revisit the role of peptides in alloreactive T cell recognition. We first compared Ab and TCR recognition of KbW9 and Kb. Six of six Kb-specific mAbs, directed against different parts of the molecule, recognized KbW9 well, albeit at different levels than Kb. Furthermore, KbW9 readily served as a restriction element for a peptide-specific syngeneic CTL response. Therefore, KbW9 mutation did not result in gross distortions of the TCR-interacting surface of class I, which was comparable between Kb and KbW9. Interestingly, when KbW9 was used to stimulate allogeneic T cells, it induced an infrequent CTL population that cross-reacted against Kb and was specific for peptide-independent MHC epitopes. By contrast, Kb-induced alloreactive CTLs recognized Kb in a peptide-specific manner, did not cross-react on KbW9, and were present at much higher frequencies than those induced by KbW9. Thus, induction of rare peptide-independent CTLs depended on unique structural features of KbW9, likely due to the elevated floor of the peptide-binding groove and the consequent protruding position of the peptide. These results shed new light on the relationship between TCR and peptide-MHC complex in peptide-independent allorecognition.
DNA Methylation Changes at Human Th2 Cytokine Genes Coincide with DNase I Hypersensitive Site Formation During CD4+ T Cell Differentiation
Authors: Santangelo Samantha , Cousins David J. , Winkelmann Nicole E. E. , Staynov Dontcho Z. 
Published: April 2014

AbstractThe differentiation of naive CD4+ T lymphocytes into Th1 and Th2 lineages generates either cellular or humoral immune responses. Th2 cells express the cytokines IL-4, -5, and -13, which are implicated in asthma and atopy. Much has been published about the regulation of murine Th2 cytokine expression, but studies in human primary T cells are less common. We have developed a method for differentiating human CD45RA+ (naive) T cells into Th1 and Th2 populations that display distinct cytokine expression profiles. We examined both CpG methylation, using bisulfite DNA modification and sequencing, and chromatin structure around the IL-4 and IL-13 genes before and after human T cell differentiation and in normal human skin fibroblasts. In naive cells, the DNA was predominantly methylated. After Th2 differentiation, DNase I hypersensitive sites (DHS) appeared at IL-4 and IL-13 and CpG demethylation occurred only around the Th2-specific DHS. Both DHS and CpG demethylation coincided with consensus binding sites for the Th2-specific transcription factor GATA-3. Although fibroblasts, like naive and Th1 cells, did not express IL-4 or IL-13, DHS and unmethylated CpG sites that were distinct from the Th2-specific sites were observed, suggesting that chromatin structure in this cluster not only varies in T cells according to IL-4/IL-13 expression but is also tissue specific.
Early Events of TCR Signaling Are Distinct in Human Th1 and Th2 Cells
Authors: Hannier Sigrid , Bitegye Christina , Demotz Stéphane 
Published: April 2014

AbstractTo study the requirements for activation of human Th1 and Th2 cells, soluble peptide/DR1 complexes were prepared from naturally expressed DR1 protein. When immobilized, this material induced T cell activation, as revealed by CD25 up-regulation. Unexpectedly, Th2 cells required a higher density of peptide/DR1 complexes than Th1 cells to initiate CD25 up-regulation. Similar findings were obtained with immobilized or soluble and cross-linked anti-CD3 mAb. In contrast, peptide/DR1 complexes displayed on the surface of nonprofessional APC similarly induced CD25 up-regulation in Th1 and Th2 cells. Signaling events distinguishing human Th1 and Th2 cells following TCR engagement by anti-CD3 mAb were then studied. It was observed that upon TCR triggering, the overall tyrosine phosphorylation profiles were fainter in Th2 than in Th1 clones. Similar results were obtained with Th1- and Th2-polarized polyclonal lines. Varying the dose of anti-CD3 mAb, the kinetics of activation, and coengagement of CD3 and CD28 failed to increase tyrosine phosphorylation in Th2 cells to levels reached in Th1 cells. In contrast, treatment with the tyrosine phosphatase inhibitor phenylarsine oxide resulted in similar tyrosine phosphorylation levels in Th2 and Th1 cells. These findings indicated that Th2 cells had an intrinsically lower TCR-induced tyrosine phosphorylation capacity than Th1 cells, which might be controlled by Th1- and Th2-specific phosphatase profiles. Finally, a weaker association was found between ZAP-70 and CD3ζ in Th2 than in Th1 cells after TCR engagement. Taken together, these results constituted evidence that early events in the TCR signaling cascades are distinct in human Th1 and Th2 cells.
Transcriptional Regulation of the Human Polymeric Ig Receptor Gene: Analysis of Basal Promoter Elements
Authors: Hempen Paula M. , Phillips Kimberly M. , Conway Pamela S. , Sandoval Katrina H. , Schneeman Tracey A. , Wu Hsin-Jung , Kaetzel Charlotte S. 
Published: April 2014

AbstractSecretory Igs provide the first line of adaptive immune defense against ingested, inhaled, and sexually transmitted pathogens at mucosal surfaces. The polymeric Ig receptor regulates transport of dimeric IgA and pentameric IgM into external secretions. The level of expression of polymeric Ig receptor is controlled to a large extent by transcription of the PIGR gene in mucosal epithelial cells. Here we present a detailed analysis of the promoter of the PIGR gene by transient transfection of luciferase reporter plasmids into cultured cell lines. Comparisons of the human and mouse PIGR promoters in human and mouse intestinal and liver cell lines demonstrated that the human PIGR promoter was 4- to 5-fold more active than the mouse PIGR promoter in all cell types, and that both the human and mouse PIGR promoters were more active in intestinal than in liver cell lines. Targeted deletions of 22-bp segments of the human PIGR promoter revealed that the region from nt −63 to −84 is crucial for basal transcription, and that two upstream regions can act as positive or negative regulators. Point mutations within the region from nt −63 to −84 demonstrated that an E box motif, which binds the basic helix-loop-helix protein upstream stimulatory factor, is required for PIGR promoter activity. Two additional regulatory motifs were identified in the proximal promoter region: a binding site for AP2, and an inverted repeat motif that binds an unidentified protein. These findings suggest that cooperative binding of multiple transcription factors regulates basal activity of the human PIGR promoter.
Repression of AP-1 Function: A Mechanism for the Regulation of Blimp-1 Expression and B Lymphocyte Differentiation by the B Cell Lymphoma-6 Protooncogene
Authors: Vasanwala Farha H. , Kusam Saritha , Toney Lisa M. , Dent Alexander L. 
Published: April 2014

Abstract
The B cell lymphoma-6 (BCL-6) transcriptional repressor protein is an important regulator of B cell differentiation and is strongly implicated in the development of B cell lymphoma. Expression of the Blimp-1 transcription factor, which is critical for promoting B cell differentiation into plasma cells, is repressed by BCL-6. We have investigated the mechanism for how BCL-6 represses Blimp-1 transcription, and have found that BCL-6 regulates the Blimp-1 promoter through a novel mechanism involving AP-1 elements. Specifically, BCL-6 is a potent repressor of transcriptional activity mediated by AP-1 factors. We found that the zinc-finger region of BCL-6 interacts with c-Jun, JunB, and JunD proteins but does not bind c-Fos or Fra-2 proteins. An estrogen receptor ligand binding domain fusion with the BCL-6 zinc finger domain can act as a estrogen-inducible dominant negative protein and increase AP-1 activity in BCL-6+ cells but not in BCL-6− cells, indicating that endogenous BCL-6 represses AP-1 activity. Additionally, we have confirmed a specific interaction between c-Jun and the zinc finger domain of BCL-6 in vivo using a mammalian two-hybrid assay. Repression of AP-1 function by BCL-6 may be a key mechanism for how BCL-6 regulates gene expression to control inflammation, lymphocyte differentiation, and lymphomagenesis.
Induction of Central Deletional T Cell Tolerance by Gene Therapy
Authors: Kang Eun-Suk , Iacomini John 
Published: April 2014

Abstract
Transgenic mice expressing an alloreactive TCR specific for the MHC class I Ag Kb were used to examine the mechanism by which genetic engineering of bone marrow induces T cell tolerance. Reconstitution of lethally irradiated mice with bone marrow infected with retroviruses carrying the MHC class I gene H-2Kb resulted in lifelong expression of Kb on bone marrow-derived cells. While CD8 T cells expressing the transgenic TCR developed in control mice reconstituted with mock-transduced bone marrow, CD8 T cells expressing the transgenic TCR failed to develop in mice reconstituted with H-2Kb transduced bone marrow. Analysis of transgene-expressing CD8 T cells in the thymus and periphery of reconstituted mice revealed that CD8 T cells expressing the transgenic TCR underwent negative selection in the thymus of mice reconstituted with Kb transduced bone marrow. Negative selection induced by gene therapy resulted in tolerance to Kb. Thus, genetic engineering of bone marrow can be used to alter T cell education in the thymus by inducing negative selection.
A Novel Functional Class I Lineage in Zebrafish (Danio rerio), Carp (Cyprinus carpio), and Large Barbus (Barbus intermedius) Showing an Unusual Conservation of the Peptide Binding Domains
Authors: Kruiswijk Corine P. , Hermsen Trudi T. , Westphal Adrie H. , Savelkoul Huub F. J. , Stet René J. M. 
Published: April 2014

AbstractSpecies from all major jawed vertebrate taxa possess linked polymorphic class I and II genes located in an MHC. The bony fish are exceptional with class I and II genes located on different linkage groups. Zebrafish (Danio rerio), common carp (Cyprinus carpio), and barbus (Barbus intermedius) represent highly divergent cyprinid genera. The genera Danio and Cyprinus diverged 50 million years ago, while Cyprinus and Barbus separated 30 million years ago. In this study, we report the first complete protein-coding class I ZE lineage cDNA sequences with high similarity between the three cyprinid species. Two unique complete protein-coding cDNA sequences were isolated in zebrafish, Dare-ZE*0101 and Dare-ZE*0102, one in common carp, Cyca-ZE*0101, and six in barbus, Bain-ZE*0101, Bain-ZE*0102, Bain-ZE*0201, Bain-ZE*0301, Bain-ZE*0401, and Bain-ZE*0402. Deduced amino acid sequences indicate that these sequences encode bonafide class I proteins. In addition, the presence of conserved potential peptide anchoring residues, exon-intron organization, ubiquitous expression, and polymorphism generated by positive selection on putative peptide binding residues support a classical nature of class I ZE lineage genes. Phylogenetic analyses revealed clustering of the ZE lineage clade with nonclassical cyprinid class I Z lineage clade away from classical cyprinid class I genes, suggesting a common ancestor of these nonclassical genes as observed for mammalian class I genes. Data strongly support the classical nature of these ZE lineage genes that evolved in a trans-species fashion with lineages being maintained for up to 100 million years as estimated by divergence time calculations.
Role That Each NKG2A Immunoreceptor Tyrosine-Based Inhibitory Motif Plays in Mediating the Human CD94/NKG2A Inhibitory Signal
Authors: Kabat Juraj , Borrego Francisco , Brooks Andrew , Coligan John E. 
Published: April 2014

AbstractThe human NKG2A chain of the CD94/NKG2A receptor contains two immunoreceptor Tyr-based inhibitory motifs (ITIMs) in its cytoplasmic tail. To determine the relative importance of membrane-distal (residues 6–11) and membrane-proximal (residues 38–43) ITIMs in mediating the inhibitory signal, we made site-directed mutants of NKG2A at the Y (Y8F, Y40F, Y8F/Y40F) and the residues two positions N-terminal (Y-2) of Y (V6A, I38A, V6A/I38A) in each motif. Wild-type (wt) and mutated NKG2A were then cotransfected with CD94 into rat basophilic leukemia 2H3 cells. Immunochemical analyses after pervanadate treatment showed that each of the mutant molecules could be phosphorylated to expected levels relative to wt NKG2A and that all the mutations significantly reduced the avidity of SH2 domain-bearing tyrosine phosphatase-1 for NKG2A. Confocal microscopy was used to determine whether SH2 domain-bearing tyrosine phosphatase-1 and CD94/NKG2A colocalized intracellularly after receptor ligation. Only the Y8F/Y40F and Y8F mutant NKG2A molecules failed to show a dramatic colocalization. In agreement with this result, the Y8F/Y40F mutant was unable to inhibit FcεRI-mediated serotonin release and the Y8F mutant was relatively ineffective compared with wt NKG2A. In contrast, the Y40F mutant was 70% as effective as wt in mediating inhibition, and the Y-2 mutations did not remarkably affect inhibitory function. These results show that, like KIR, both NKG2A ITIMs are required for mediating the maximal inhibitory signal, but opposite to KIR, the membrane-distal ITIM is of primary importance rather than the membrane-proximal ITIM. This probably reflects the opposite orientation of the ITIMs in type II vs type I proteins.
Lipid Microdomains Are Required Sites for the Selective Endocytosis and Nuclear Translocation of IFN-γ, Its Receptor Chain IFN-γ Receptor-1, and the Phosphorylation and Nuclear Translocation of STAT1α
Authors: Subramaniam Prem S. , Johnson Howard M. 
Published: April 2014

AbstractIFN-γ contains a nuclear localization sequence that may play a role in the nuclear transport of activated STAT1α via a complex of IFN-γ/IFN-γ receptor (IFNGR)-1/STAT1α with the nuclear importer nucleoprotein interactor 1. In this study, we examine the mechanism of endocytosis of IFNGR-1 and the relationship of its nuclear translocation to that of STAT1α. In untreated WISH cells, both IFNGR-1 and IFNGR-2 were constitutively localized within caveolae-like microdomains isolated from plasma membrane. However, treatment of cells with IFN-γ resulted in rapid migration of IFNGR-1, but not IFNGR-2, from these microdomains. Filipin pretreatment, which specifically inhibits endocytosis from caveolae-like microdomains, inhibited the nuclear translocation of IFN-γ and IFNGR-1 as well as the tyrosine phosphorylation and nuclear translocation of STAT1α, but did not affect the binding of IFN-γ to these cells. In the Jurkat T lymphocyte cell line, which does not express caveolin-1, nuclear translocation of IFNGR-1 and STAT1α were similarly inhibited by filipin pretreatment. Isolation of lipid microdomains from Jurkat cells showed that both IFNGR-1 and IFNGR-2 were associated with lipid microdomains only after stimulation with IFN-γ, suggesting that the IFNGR subunits are recruited to lipid microdomains by IFN-γ binding in lymphocytes (Jurkat) in contrast to their constitutive presence in epithelial (WISH) cells. In contrast, treatments that block clathrin-dependent endocytosis did not inhibit either activation or nuclear translocation of STAT1α or the nuclear translocation of IFN-γ or IFNGR-1. Thus, membrane lipid microdomains play an important role in IFN-γ-initiated endocytic events involving IFNGR-1, and the nuclear translocation of IFN-γ, IFNGR-1, and STAT1α.
Detection of Functional VH81X Heavy Chains in Adult Mice with an Assessment of Complementarity-Determining Region 3 Diversity and Capacity to Form Pre-B Cell Receptor
Authors: Hayden Tracy A. , Riegert Patricia , Kline Gregory H. 
Published: April 2014

AbstractRecent reports have indicated that up to 50% of all H chain proteins formed cannot associate with the surrogate L chain complex and therefore fail to form a pre-B cell receptor (pBCR), which is required for allelic exclusion and, in most cases, verifies that the H chain can assemble with the L chain to form an Ab molecule. Certain VH genes, such as VH81X, appear to be particularly prone to encoding for nonpairing (dysfunctional) H chains. It has been suggested that sequence variability at complementarity-determining region 3, especially when increased by the enzyme TdT, often precludes the ability of VH81X-using H chains to form pBCR. To determine whether a motif exists that accounts for the ability of H chains to pair with surrogate L chain complex/L chain, we have bred a mouse line in which H chain recombination can only occur on one allele, allowing us to compile a pool of H chains capable of forming Ab molecules in the absence of dysfunctional H chains. Somewhat unexpectedly, we have found VH81X H chains capable of Ab formation and cell surface expression in the presence of TdT. Scrutiny of these H chains has revealed that, although highly prone to encode for dysfunctional H chains, sequence variability is not severely limited among functional VH81X H chains. We also demonstrate that surface Ig expression is highly indicative of the capacity of a H chain to form pBCR.