bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipid Microdomain Clustering Induces a Redistribution of Antigen Recognition and Adhesion Molecules on Human T Lymphocytes
Authors: Mitchell Jason S. , Kanca Oguz , McIntyre Bradley W.
Published: April 2014

The study of lipid microdomains in the plasma membrane is a topic of recent interest in leukocyte biology. Many T cell activation and signaling molecules are found to be associated with lipid microdomains and have been implicated in normal T cell function. It has been proposed that lipid microdomains with their associated molecules move by lateral diffusion to areas of cellular interactions to initiate signaling pathways. Using sucrose density gradients we have found that human T cell β1 integrins are not normally associated with lipid microdomains. However, cross-linking of GM1 through cholera toxin B-subunit (CTB) causes an enrichment of β1 integrins in microdomain fractions, suggesting that cross-linking lipid microdomains causes a reorganization of molecular associations. Fluorescent microscopy was used to examine the localization of various lymphocyte surface molecules before and after lipid microdomain cross-linking. Lymphocytes treated with FITC-CTB reveal an endocytic vesicle that is enriched in TCR and CD59, while β1 integrin, CD43, and LFA-3 were not localized in the vesicle. However, when anti-CTB Abs are used to cross-link lipid microdomains, the microdomains are not internalized but are clustered on the cell surface. In this study, CD59, CD43, and β1 integrin are all seen to colocalize in a new lipid microdomain from which LFA-3 remains excluded and the TCR is now dissociated. These findings show that cross-linking lipid microdomains can cause a dynamic rearrangement of the normal order of T lymphocyte microdomains into an organization where novel associations are created and signaling pathways may be initiated.
HIV-1 Down-Modulates γ Signaling Chain of FcγR in Human Macrophages: A Possible Mechanism for Inhibition of Phagocytosis
Authors: Kedzierska Katherine , Ellery Philip , Mak Johnson , Lewin Sharon R. , Crowe Suzanne M. , Jaworowski Anthony
Published: April 2014

HIV-1 infection impairs a number of macrophage effector functions, thereby contributing to development of opportunistic infections and the pathogenesis of AIDS. FcγR-mediated phagocytosis by human monocyte-derived macrophages (MDM) is inhibited by HIV-1 infection in vitro, and the underlying mechanism was investigated in this study. Inhibition of phagocytosis directly correlated with the multiplicity of HIV-1 infection. Expression of surface FcγRs was unaffected by HIV-1 infection, suggesting that inhibition of phagocytosis occurred during or after receptor binding. HIV-1 infection of MDM markedly inhibited tyrosine phosphorylation of the cellular proteins, which occurs following engagement of FcγRs, suggesting a defect downstream of initial receptor activation. FcγR-mediated phagocytosis in HIV-infected MDM was associated with inhibition of phosphorylation of tyrosine kinases from two different families, Hck and Syk, defective formation of Syk complexes with other tyrosine-phosphorylated proteins, and inhibition of paxillin activation. Down-modulation of protein expression but not mRNA of the γ signaling subunit of FcγR (a docking site for Syk) was observed in HIV-infected MDM. Infection of MDM with a construct of HIV-1 in which nef was replaced with the gene for the γ signaling subunit augmented FcγR-mediated phagocytosis, suggesting that down-modulation of γ-chain protein expression in HIV-infected MDM caused the defective FcγR-mediated signaling and impairment of phagocytosis. This study is the first to demonstrate a specific alteration in phagocytosis signal transduction pathway, which provides a mechanism for the observed impaired FcγR-mediated phagocytosis in HIV-infected macrophages and contributes to the understanding of how HIV-1 impairs cell-mediated immunity leading to HIV-1 disease progression.
A 320-Kilobase Artificial Chromosome Encoding the Human HLA DR3-DQ2 MHC Haplotype Confers HLA Restriction in Transgenic Mice
Authors: Chen Zhenjun , Dudek Nadine , Wijburg Odilia , Strugnell Richard , Brown Lorena , Deliyannis Georgia , Jackson David , Koentgen Frank , Gordon Tom , McCluskey James
Published: April 2014

MHC class II haplotypes control the specificity of Th immune responses and susceptibility to many autoimmune diseases. Understanding the role of HLA class II haplotypes in immunity is hampered by the lack of animal models expressing these genes as authentic cis-haplotypes. In this study we describe transgenic expression of the autoimmune prone HLA DR3-DQ2 haplotype from a yeast artificial chromosome (YAC) containing an intact ∼320-kb region from HLA DRA to DQB2. In YAC-transgenic mice HLA DR and DQ gene products were expressed on B cells, macrophages, and dendritic cells, but not on T cells indicating cell-specific regulation. Positive selection of the CD4 compartment by human class II molecules was 67% efficient in YAC-homozygous mice lacking endogenous class II molecules (Aβnull/null) and expressing only murine CD4. A broad range of TCR Vβ families was used in the peripheral T cell repertoire, which was also purged of Vβ5-, Vβ11-, and Vβ12-bearing T cells by endogenous mouse mammary tumor virus-encoded superantigens. Expression of the HLA DR3-DQ2 haplotype on the Aβnull/null background was associated with normal CD8-dependent clearance of virus from influenza-infected mice and development of CD4-dependent protection from otherwise lethal infection with Salmonella typhimurium. HLA DR- and DQ-restricted T cell responses were also elicited following immunization with known T cell determinants presented by these molecules. These findings demonstrate the potential for human MHC class II haplotypes to function efficiently in transgenic mice and should provide valuable tools for developing humanized models of MHC-associated autoimmune diseases.
Cutting Edge: Differential Segregation of the Src Homology 2-Containing Protein Tyrosine Phosphatase-1 Within the Early NK Cell Immune Synapse Distinguishes Noncytolytic from Cytolytic Interactions
Authors: Vyas Yatin M. , Maniar Hina , Dupont Bo
Published: April 2014

Inhibitory NK receptors with ligand specificity for MHC class I recruit Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) phosphatase and prevent autocytotoxicity. Activation of SHP-1 depends upon Src kinase-mediated tyrosine phosphorylation of the cytoplasmic domain of the inhibitory receptor. In this study we demonstrate, by quantitative temporal analysis, that talin, Lck, and SHP-1 are recruited to the synapse within 1 min in both cytolytic and noncytolytic conjugates. Polarization of talin and Lck rapidly disappears in the noncytolytic interactions but persists in cytolytic interactions, where protein kinase C-θ, Src homology 2 domain-containing leukocyte protein of 76 kDa, and lysosomes are recruited within 5 min. At 1 min SHP-1 clusters in the periphery of the cytolytic synapse, whereas it clusters in the center of the noncytolytic synapse. Lck has multifocal distribution in both synapses consistent with the shared requirement for early tyrosine phosphorylation. Our studies indicate that the spatial location of SHP-1 in the synapse distinguishes noncytolytic from cytolytic interactions within the first minute.
Interaction Between Src Homology 2 Domain Bearing Protein Tyrosine Phosphatase Substrate-1 and CD47 Mediates the Adhesion of Human B Lymphocytes to Nonactivated Endothelial Cells
Authors: Yoshida Hitoshi , Tomiyama Yoshiaki , Oritani Kenji , Murayama Yoko , Ishikawa Jun , Kato Hisashi , Miyagawa Jun-ichiro , Honma Nakayuki , Nishiura Tetsuo , Matsuzawa Yuji
Published: April 2014

CD47 modulates a variety of cell functions such as adhesion, spreading, and migration. Using a fusion protein consisting of the extracellular region of Src homology 2 domain bearing protein tyrosine phosphatase substrate-1 (SHPS-1) and the Fc portion of human Ig (SHPS-1-Ig) we investigated the effects of SHPS-1 as a ligand for CD47 on B lymphocytes. Although SHPS-1-Ig binding to human B cell lines was solely mediated via CD47, their binding capacity for soluble and immobilized SHPS-1-Ig varied among cell lines irrespective of the similar expression levels of CD47, suggesting that distinctive affinity/avidity states exist during B cell maturation. Nalm6 cell line and tonsilar B lymphocytes adhered to immobilized SHPS-1-Ig and showed polarization-like morphology. These effects of SHPS-1-Ig were blocked by anti-CD47 mAbs (B6H12 and SE5A5). Wortmannin, a phosphatidylinositol-3 kinase inhibitor, but not pertussis toxin significantly inhibited the polarization induced by the immobilized SHPS-1-Ig. Thus, SHPS-1 acts as an adhesive substrate via CD47 in human B lymphocyte. Immunohistochemical analyses indicated that SHPS-1 is expressed on high endothelial venule as well as macrophages in human tonsils. HUVECs also express SHPS-1 in the absence of any stimuli, and the adhesion of tonsilar B lymphocytes to nonactivated HUVECs was significantly inhibited by SE5A5, indicating that SHPS-1/CD47 interaction is involved in the adhesion. Our findings suggest that SHPS-1/CD47 interaction may contribute to the recruitment of B lymphocytes via endothelial cells under steady state conditions.
Expression of Inhibitory Receptors Ly49E and CD94/NKG2 on Fetal Thymic and Adult Epidermal TCR Vγ3 Lymphocytes
Authors: Van Beneden Katrien , De Creus An , Stevenaert Frederik , Debacker Veronique , Plum Jean , Leclercq Georges
Published: April 2014

Ly49 and CD94/NKG2 inhibitory receptors are predominantly expressed on murine NK cells, but they are also expressed on a subpopulation of peripheral CD8 memory TCR αβ lymphocytes. In this study we demonstrate that Ly49E and CD94/NKG2 receptors are expressed on mature TCR Vγ3+ cells in the fetal thymus. Expression correlated with a memory phenotype, such as expression of CD44, 2B4, and IL-2Rβ (CD122), and absence of IL-2Rα (CD25) expression. No expression of Ly49A, C, D, G2, or I receptors was observed. This phenotype is similar to that of fetal thymic NK cells. Skin-located Vγ3 T cells, the progeny of fetal thymic Vγ3 cells, also expressed CD94/NKG2 and Ly49E but not the other members of the Ly49 family. The development and survival of Ly49E+ or CD94/NKG2+ Vγ3 T lymphocytes was not dependent upon expression of MHC class I molecules. The cytotoxicity of TCR Vγ3 cells was inhibited when Qdm, the ligand for CD94/NKG2, was presented by Qa1b-transfected target cells. Also, upon cross-linking of CD94/NKG2 with mAb 3S9, TCR Vγ3 thymocytes were prevented from killing FcγR+ P815 target cells. These effects were most pronounced in the CD94/NKG2high subpopulation as compared with the CD94/NKG2low subpopulation of Vγ3 cells. Our data demonstrate that Vγ3 T cells expressing inhibitory Ly49E and CD94/NKG2 receptors are mature and display a memory phenotype, and that CD94/NKG2 functions as an inhibitory receptor on these T lymphocytes.
The First Cytokine Sequence Within Cartilaginous Fish: IL-1β in the Small Spotted Catshark (Scyliorhinus canicula)
Authors: Bird Steve , Wang Tiehui , Zou Jun , Cunningham Charlie , Secombes Chris J.
Published: April 2014

Cartilaginous fish are considered the most primitive living jawed vertebrates with a complex immune system typical of all jawed vertebrates. Cytokine homologs are found within jawless and bony fish, although no cytokine or cytokine receptor genes have been sequenced in cartilaginous fish. In this study the complete coding sequence of the small spotted catshark (Scyliorhinus canicula) IL-1β gene is presented that contains a short 5′ untranslated region (54 bp), a 903-bp open reading frame, a 379-bp 3′ untranslated region, a polyadenylation signal, and eight mRNA instability motifs. The predicted translation (301 amino acids) has highest identity to trout IL-1β (31.7%), with greatest homology within the putative 12 β-sheets. The IL-1 family signature is also present, but there is no apparent signal peptide. As with other nonmammalian IL-1β sequences, the IL-1-converting enzyme cut site is absent. Expression of the IL-1β transcript is detectable by RT-PCR in the spleen and testes, induced in vivo with LPS. Furthermore, a 7-fold increase of transcript levels in splenocytes incubated for 5 h with LPS was seen. The genomic organization comprises six exons and five introns with highest homology seen in exons encoding the largest amount of secondary structure per amino acid. Southern blot analysis suggests at least two copies of the IL-1β gene or genes related to the 3′ end of the IL-1β sequence are present in the catshark. The cloning of IL-1β in S. canicula, the first cytokine sequenced within cartilaginous fish, verifies previous bioactivity evidence for the presence of inflammatory cytokines.
Functional Analysis of Iα Promoter Regions of Multiple IgA Heavy Chain Genes
Authors: Spieker-Polet Helga , Yam Pi-Chen , Knight Katherine L.
Published: April 2014

The 13 nonallelic IgA H chain genes of rabbit are differentially expressed in vivo. They can be grouped into those expressed at high levels (Cα4, Cα5, Cα6, Cα9, Cα10, Cα12, and Cα13), those expressed at low levels (Cα1, Cα2, Cα7, and Cα11), and those that are not expressed (Cα3 and Cα8). We tested whether the differential in vivo expression is due to differential responses of the Iα promoters to TGF-β stimulation. We stimulated the rabbit B cell line 55D1 with TGF-β and, using single-cell RT-PCR, found that expression of germline (GL) transcripts of α3 and α8 could not be induced. By luciferase reporter gene assay and EMSA we found that the promoters of the unexpressed isotypes Cα3 and Cα8 are defective, thereby explaining the absence of IgA3 and IgA8 in vivo. When comparing the promoter activities of the other isotypes we found that the activities did not reflect the degree of in vivo expression. Instead, the promoters of the isotypes expressed at high or low levels promoted expression of the luciferase gene to a similar degree, except for the Iα4 promoter, which had much higher activity. Also the degree to which TGF-β induced GL expression of the various isotypes in 55D1 B cells did not reflect in vivo expression. However, most of the TGF-β-stimulated cells expressed GL mRNA of multiple isotypes; no isotype was expressed preferentially. These results suggest that the final switch to a single isotype is regulated in a step subsequent to GL transcription, rather than by induction of GL transcripts by the Iα promoter.
Segregation of Bad from Lipid Rafts Is Implicated in the Induction of Apoptosis
Authors: Ayllón Verónica , Fleischer Aarne , Cayla Xavier , García Alphonse , Rebollo Angelita
Published: April 2014

Many molecules relocate subcellularly in cells undergoing apoptosis. Using coimmunoprecipitation experiments we demonstrate that Bad is not associated to 14-3-3 protein, suggesting a new mechanism for the control of the proapoptotic role of Bad. Here we show, by confocal microscopy and cellular fractionation, that Bad is attached to lipid rafts in IL-4-stimulated cells and thymocytes while associated with mitochondria in IL-4-deprived cells. Disruption of lipid rafts by methyl-β-cyclodextrin treatment induces segregation of Bad from rafts, which correlates with apoptosis. Our results suggest that the interaction of Bad with rafts is a dynamic process regulated by IL-4 and involved in the control of apoptosis.
Antibody Efficacy in Murine PulmonaryCryptococcus neoformansInfection: A Role for Nitric Oxide
Authors: Rivera Johanna , Mukherjee Jean , Weiss Louis M. , Casadevall Arturo
Published: April 2014

We investigated the pathogenesis of pulmonary Cryptococcus neoformans infection and passive Ab efficacy in mice deficient in inducible NO synthase (NOS2−/−) and the parental strain. Parental mice lived significantly longer than NOS2−/− mice after intratracheal infection, despite having a higher lung fungal burden. Administration of Ab reduced lung CFU in both NOS2−/− and parental mice, but prolonged survival and increased the inflammatory response only in parental mice. Ab administration was associated with increased serum nitrite and reduced polysaccharide levels in parental mice. Eosinophils were present in greater numbers in the lung of infected NOS2−/− mice than parental mice, irrespective of Ab administration. C. neoformans infection in NOS2−/− mice resulted in significantly higher levels of IFN-γ, monocyte chemoattractant protein-1, and macrophage-inflammatory protein-1α than parental mice. Ab administration had different effects on infected NOS2−/− and parental mice with respect to IFN-γ, monocoyte chemoattractant protein-1, and macrophage-inflammatory protein-1α levels. Ab administration increased lung levels of IFN-γ in parental mice and reduced levels in NOS2−/− mice. The results indicate that NO is involved in the regulation of cytokine expression in response to cryptococcal pneumonia and is necessary for Ab efficacy against C. neoformans in mice. Our findings indicate a complex relationship between Ab efficacy against C. neoformans and cytokine expression, underscoring the interdependency of cellular and humoral defense mechanisms.
The Proinflammatory CD14+CD16+DR++ Monocytes Are a Major Source of TNF
Authors: Belge Kai-Uwe , Dayyani Farshid , Horelt Alexia , Siedlar Maciej , Frankenberger Marion , Frankenberger Bernhard , Espevik Terje , Ziegler-Heitbrock Löms
Published: April 2014

In human blood two monocyte populations can be distinguished, i.e., the CD14++CD16−DR+ classical monocytes and the CD14+CD16+DR++ proinflammatory monocytes that account for only 10% of all monocytes. We have studied TNF production in these two types of cells using three-color immunofluorescence and flow cytometry on whole peripheral blood samples stimulated with either LPS or with the bacterial lipopeptide S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys4-OH,trihydrochloride (Pam3Cys). After stimulation with LPS the median fluorescence intensity for TNF protein was 3-fold higher in the proinflammatory monocytes when compared with the classical monocytes. After stimulation with Pam3Cys they almost exclusively responded showing 10-fold-higher levels of median fluorescence intensity for TNF protein. The median fluorescence intensity for Toll-like receptor 2 cell surface protein was found 2-fold higher on CD14+CD16+DR++ monocytes, which may explain, in part, the higher Pam3Cys-induced TNF production by these cells. When analyzing secretion of TNF protein into the supernatant in PBMCs after depletion of CD16+ monocytes we found a reduction of LPS-induced TNF by 28% but Pam3Cys-induced TNF was reduced by 64%. This indicates that the minor population of CD14+CD16+ monocytes are major producers of TNF in human blood.
IL-18-Binding Protein Expression by Endothelial Cells and Macrophages Is Up-Regulated During Active Crohn’s Disease
Authors: Corbaz Anne , ten Hove Tessa , Herren Suzanne , Graber Pierre , Schwartsburd Boris , Belzer Ilana , Harrison Jillian , Plitz Thomas , Kosco-Vilbois Marie H. , Kim Soo-Hyun , Dinarello Charles A. , Novick Daniela , van Deventer Sander , Chvatchko Yolande
Published: April 2014

The pathogenesis of Crohn’s disease (CD) remains under intense investigation. Increasing evidence suggests a role for mature IL-18 in the induction of proinflammatory cytokines and Th1 polarization in CD lesions. The aim of this study was to investigate the contribution of the IL-18-neutralizing (a and c) and non-neutralizing (b and d) isoforms of IL-18-binding protein (IL-18BP) during active CD. Intestinal endothelial cells and macrophages were the major source of IL-18BP within the submucosa, and this IL-18BP production was also found to be relevant to other types of endothelial cells (HUVEC) and macrophages (peripheral monocytes). IL-18BP messenger transcript and protein were significantly increased in surgically resected specimens from active CD compared with control patients, correlating with an up-regulation of IL-18. Analysis of the expression of the four IL-18BP isoforms as well as being free or bound to IL-18 was reported and revealed that unbound IL-18BP isoforms a and c and inactive isoform d were present in specimens from active CD and control patients while isoform b was not detected. IL-18/IL-18BP complex was also detected. Interestingly, although most was complexed, free mature IL-18 could still be detected in active CD specimens even in the presence of the IL-18BP isoform a/c. These results demonstrate that the appropriate neutralizing isoforms are present in the intestinal tissue of patients with active CD and highlights the complexity of IL-18/IL-18BP biology.
Human DQ8 Can Substitute for Murine I-Ag7 in the Selection of Diabetogenic T Cells Restricted to I-Ag71
Authors: Wen Li , Wong F. Susan , Sherwin Robert , Mora Conchi
Published: April 2014

The strong association of type 1 diabetes with specific MHC class II genes, such as I-Ag7 in nonobese diabetic mice and HLA-DQ8 in humans, suggests that MHC class II molecules play an important role in the development of the disease. To test whether human DQ8 molecules could cross the species barrier and functionally replace their murine homolog I-Ag7, we generated DQ8/BDC2.5 transgenic mice. We have shown that BDC2.5 transgenic T cells are selected on DQ8 in the thymus and cause diabetes in a manner similar to that seen when the T cells are selected on H2g7. Splenocytes from DQ8/BDC2.5 mice also showed reactivity toward islets in vitro as seen in H-2g7/BDC2.5 mice. We conclude that DQ8 molecules not only share structural similarity with the murine homolog I-Ag7, but also can cross the species barrier and functionally replace I-Ag7 molecules to stimulate diabetogenic T cells and produce diabetes.
Dynamics of T Cell Responses in HIV Infection
Authors: Appay Victor , Papagno Laura , Spina Celsa A. , Hansasuta Pokrath , King Abigail , Jones Louise , Ogg Graham S. , Little Susan , McMichael Andrew J. , Richman Douglas D. , Rowland-Jones Sarah L.
Published: April 2014

Cytotoxic CD8+ T cells play a major role in the immune response against viruses. However, the dynamics of CD8+ T cell responses during the course of a human infection are not well understood. Using tetrameric complexes in combination with a range of intracellular and extracellular markers, we present a detailed analysis of the changes in activation and differentiation undergone by Ag-specific CD8+ T cells, in relation to Ag-specific CD4+ T cell responses, in the context of a human infection: HIV-1. During primary HIV-1 infection, the initial population of HIV-specific CD8+ T cells is highly activated and prone to apoptosis. The Ag-specific cells differentiate rapidly from naive to cells at a perforin low intermediate stage of differentiation, later forming a stable pool of resting cells as viral load decreases during chronic infection. These observations have significant implications for our understanding of T cell responses in human viral infections in general and indicate that the definition of effector and memory subsets in humans may need revision.
A Ligand for the Murine NK Activation Receptor Ly-49D: Activation of Tolerized NK Cells from β2-Microglobulin- Deficient Mice
Authors: Furukawa Hiroshi , Iizuka Koho , Poursine-Laurent Jennifer , Shastri Nilabh , Yokoyama Wayne M.
Published: April 2014

Mouse NK cells express inhibitory NK receptors that recognize target cell MHC class I molecules and activation receptors that are less well defined. The Ly-49D activation receptor on C57BL/6 NK cells recognizes Chinese hamster ovary cells and triggers natural killing. In this study, we demonstrate that a Chinese hamster classical MHC class I molecule is the ligand for Ly-49D in a reporter gene assay system as well as in NK cell killing assays. Ly-49D recognizes the Chinese hamster class I molecule better when it is expressed with Chinese hamster β2-microglobulin (β2m) than murine β2m. However, it is still controversial that Ly-49D recognizes H-2Dd, as we were unable to demonstrate the specificity previously reported. Using this one ligand-one receptor recognition system, function of an NK activation receptor was, for the first time, investigated in NK cells that are tolerized in β2m-deficient mice. Surprisingly, Ly-49D-killing activity against ligand-expressing targets was observed with β2m-deficient mouse NK cells, albeit reduced, even though “tolerized” function of Ly-49D was expected. These results indicate that Ly-49D specifically recognizes the Chinese hamster MHC class I molecule associated with Chinese hamster β2m, and indicate that the Ly-49D NK cell activation receptor is not tolerized in β2m deficiency.
Synergistic Antitumor Effect of Bispecific CD19 × CD3 and CD19 × CD16 Diabodies in a Preclinical Model of Non-Hodgkin’s Lymphoma
Authors: Kipriyanov Sergey M. , Cochlovius Björn , Schäfer Holger J. , Moldenhauer Gerhard , Bähre Alexandra , Le Gall Fabrice , Knackmuss Stefan , Little Melvyn
Published: April 2014

To target NK cells against non-Hodgkin’s lymphoma, we constructed a bispecific diabody (BsDb) with reactivity against both human CD19 and FcγRIII (CD16). Bacterially produced CD19 × CD16 BsDb specifically interacted with both CD19+ and CD16+ cells and exhibited significantly higher apparent affinity and slower dissociation from the tumor cells than from effector cells. It was able to induce specific lysis of tumor cells in the presence of isolated human NK cells or nonfractionated PBLs. The combination of the CD19 × CD16 BsDb with a previously described CD19 × CD3 BsDb and CD28 costimulation significantly increased the lytic potential of human PBLs. Treatment of SCID mice bearing an established Burkitt’s lymphoma (5 mm in diameter) with human PBLs, CD19 × CD16 BsDb, CD19 × CD3 BsDb, and anti-CD28 mAb resulted in the complete elimination of tumors in 80% of animals. In contrast, mice receiving human PBLs in combination with either diabody alone showed only partial tumor regression. These data clearly demonstrate the synergistic effect of small recombinant bispecific molecules recruiting different populations of human effector cells to the same tumor target.
Blebs and Apoptotic Bodies Are B Cell Autoantigens
Authors: Cocca Brian A. , Cline Amy M. , Radic Marko Z.
Published: April 2014

Mounting evidence suggests that systemic lupus erythematosus autoantigens are derived from apoptotic cells. To characterize the potential interactions between apoptotic cells and B cells, the D56R/S76R variant of 3H9, a murine autoantibody that binds to DNA, chromatin, and anionic phospholipids, was compared with DNA4/1, a human anti-DNA autoantibody. Flow cytometry revealed that only D56R/S76R bound to Jurkat cells treated with either of three distinct proapoptotic stimuli, Ab binding was dependent on caspase activity, and immunoreactivity developed subsequent to annexin V binding. Confocal microscopy established a structural basis for the distinct kinetics of binding. D56R/S76R preferentially bound to membrane blebs of apoptotic cells, whereas annexin V binding did not require blebs. Inhibition of ROCK I kinase, an enzyme that stimulates nuclear fragmentation and fragment distribution into blebs, significantly reduced Ab binding. Because members of the collectin and pentraxin families of serum proteins bind to blebs on apoptotic cells and assist in the clearance of cellular remains, our results suggest that Abs to blebs could affect the recognition of apoptotic cells by cells of the innate immune system and thus modify tolerance to nuclear Ags.
Spatial Raft Coalescence Represents an Initial Step in FcγR Signaling
Authors: Kono Hajime , Suzuki Takeshi , Yamamoto Kazuhiko , Okada Masato , Yamamoto Tadashi , Honda Zen-ichiro
Published: April 2014

Characterization of lipid rafts as separated membrane microdomains consist of heterogeneous proteins suggesting that lateral assembly of rafts after Ag receptor cross-linking represents the earliest signal generating process. In line with the concept, cross-linked Ag receptors have been shown to associate with detergent-insoluble raft fraction without the aid of Src family kinases. However, it has not been established whether spatial raft coalescence could also precede Src family kinase activation. In this study, we showed that spatial raft coalescence after low-affinity FcγR cross-linking in RAW264.7 macrophages is independent of Src family kinase activity. The lateral raft assembly was found to be ascribed to the action of ligand-binding subunits, rather than to immunoreceptor tyrosine-based activation motif-bearing signal subunits, because monomeric murine FcγRIIb expressed in rat basophilic leukemia cells successfully induced spatial raft reorganization after cross-linking. We also showed that extracellular and transmembrane region of FcγRIIb is sufficient for raft stabilization. Moreover, this receptor fragment triggers rapid calcium mobilization and linker for activation of T cells phosphorylation, in a manner sensitive to Src family kinase inhibition and to cholesterol depletion. Presence of immunoreceptor tyrosine-based inhibitory motif and addition of immunoreceptor tyrosine-based activation motif to the receptor fragment abolished and enhanced the responses, respectively, but did not affect raft stabilization. These findings support the concept that ligand-binding subunit is responsible for raft coalescence, and that this event triggers initial biochemical signaling.
A Therapeutic CD4 Monoclonal Antibody Inhibits TCR-ζ Chain Phosphorylation, ζ-Associated Protein of 70-kDa Tyr319 Phosphorylation, and TCR Internalization in Primary Human T Cells
Authors: Harding Susanne , Lipp Peter , Alexander Denis R.
Published: April 2014

The molecular mechanisms mediating the inhibitory effects of a humanized CD4 mAb YHB.46 on primary human CD4+ T cells were investigated. Preincubation of T cells with soluble YHB.46 caused a general inhibition of TCR-stimulated protein tyrosine phosphorylation events, including a reduction in phosphorylation of p95vav, linker for activation of T cells, and Src homology 2 domain-containing leukocyte protein of 76-kDa signaling molecules. A marked reduction in activation of the Ras/mitogen-activated protein kinase pathway was also observed. Examination of the earliest initiation events of TCR signal transduction showed that YHB.46 inhibited TCR-ζ chain phosphorylation together with recruitment and tyrosine phosphorylation of the ζ-associated protein of 70-kDa tyrosine kinase, particularly at Tyr319, as well as reduced recruitment of p56lck to the TCR-ζ and ζ-associated protein of 70-kDa complex. These inhibitory events were associated with inhibition of TCR endocytosis. Our results show that the YHB.46 mAb is a powerful inhibitor of the early initiating events of TCR signal transduction.
Transcriptional Regulation of GATA-3 by an Intronic Regulatory Region and Fetal Liver Zinc Finger Protein 1
Authors: Hwang Eun Sook , Choi Andrew , Ho I-Cheng
Published: April 2014

GATA-3 is a T cell-specific transcription factor and is essential for the development of the T cell lineage. The transcriptional regulation of GATA-3, however, remains elusive. In this study, we report the identification of a regulatory region located within the first intron of the murine GATA-3 gene. The intronic regulatory region contains both a positive and a negative cis-acting element but, as a whole, serves as a potent T cell-specific enhancer and is essential for the promoter activity in vitro. By using yeast one-hybrid screening, we discovered that fetal liver zinc finger protein 1 (Fliz1) could bind specifically to the negative cis-acting element, the sequence of which is conserved between the mouse and human GATA-3 genes. More importantly, overexpression of Fliz1 repressed the expression of GATA-3 in vivo and in vitro. Our data suggest that the expression of GATA-3 might be partly regulated by the intronic regulatory region and Fliz1 in a developmental stage-specific fashion.
Recruitment of Phosphatidylinositol 3-Kinase to CD28 Inhibits HIV Transcription by a Tat-Dependent Mechanism
Authors: Cook Julie A. , August Avery , Henderson Andrew J.
Published: April 2014

Activation through the TCR and the costimulatory molecule CD28 influences the susceptibility of T cells to HIV-1 infection and regulates proviral gene expression. Signaling events initiated by CD28 that directly impact HIV-1 transcription have not been fully characterized. T cell lines expressing CD8α/28 chimeric receptors containing a mutation in tyrosine 173 to phenylalanine, which inhibits the recruitment of phosphatidylinositol 3-kinase (PI3K) to CD28, expressed higher levels of HIV-1 following T cell activation. Whereas constitutively active PI3K decreased provirus transcription, inhibiting endogenous PI3K with specific inhibitors or by overexpressing PTEN phosphatase enhanced HIV-1 expression. PI3K-dependent inhibition required the viral Tat protein and a trans activation response region element. Tat pull-down and coimmunoprecipitation experiments indicate that PI3K affects the formation of the Tat-associated kinase trans-activating complex. These studies demonstrate that PI3K negatively impacts HIV-1 transcription and that Tat activity is sensitive to T cell signaling events.
Unraveling of the Polymorphic Cλ2-Cλ3 Amplification and the Ke+Oz− Polymorphism in the Human Igλ Locus
Authors: van der Burg Mirjam , Barendregt Barbara H. , van Gastel-Mol Ellen J. , Tümkaya Talip , Langerak Anton W. , van Dongen Jacques J. M.
Published: April 2014

Two polymorphisms of the human Igλ (IGL) locus have been described. The first polymorphism concerns a single, 2- or 3-fold amplification of 5.4 kb of DNA in the Cλ2-Cλ3 region. The second polymorphism is the Mcg−Ke+Oz− isotype, which has only been defined via serological analyses in Bence-Jones proteins of multiple myeloma patients and was assumed to be encoded by a polymorphic Cλ2 segment because of its high homology with the Mcg−Ke−Oz− Cλ2 isotype. It has been speculated that the Mcg−Ke+Oz− isotype might be encoded by a Cλ gene segment of the amplified Cλ2-Cλ3 region. We now unraveled both IGL gene polymorphisms. The amplification polymorphism appeared to result from a duplication, triplication, or quadruplication of a functional J-Cλ2 region and is likely to have originated from unequal crossing over of the J-Cλ2 and J-Cλ3 region via a 2.2-kb homologous repeat. The amplification polymorphism was found to result in the presence of one to five extra functional J-Cλ2 per genome regions, leading to decreased Igκ:Igλ ratios on normal peripheral blood B cells. Via sequence analysis, we demonstrated that the Mcg−Ke+Oz− isotype is encoded by a polymorphic Cλ2 segment that differs from the normal Cλ2 gene segment at a single nucleotide position. This polymorphism was identified in only 1.5% (2 of 134) of individuals without J-Cλ2 amplification polymorphism and was not found in the J-Cλ2 amplification polymorphism of 44 individuals, indicating that the two IGL gene polymorphisms are not linked.
Neutrophil Transepithelial Migration: Evidence for Sequential, Contact-Dependent Signaling Events and Enhanced Paracellular Permeability Independent of Transjunctional Migration
Authors: Edens Heather A. , Levi Boaz P. , Jaye David L. , Walsh Shaun , Reaves Titus A. , Turner Jerrold R. , Nusrat Asma , Parkos Charles A.
Published: April 2014

Active migration of polymorphonuclear leukocytes (PMN) through the intestinal crypt epithelium is a hallmark of inflammatory bowel disease and correlates with patient symptoms. Previous in vitro studies have shown that PMN transepithelial migration results in increased epithelial permeability. In this study, we modeled PMN transepithelial migration across T84 monolayers and demonstrated that enhanced paracellular permeability to small solutes occurred in the absence of transepithelial migration but required both PMN contact with the epithelial cell basolateral membrane and a transepithelial chemotactic gradient. Early events that occurred before PMN entering the paracellular space included increased permeability to small solutes (<500 Da), enhanced phosphorylation of regulatory myosin L chain, and other as yet undefined proteins at the level of the tight junction. No redistribution or loss of tight junction proteins was detected in these monolayers. Late events, occurring during actual PMN transepithelial migration, included redistribution of epithelial serine-phosphorylated proteins from the cytoplasm to the nucleus in cells adjacent to migrating PMN. Changes in phosphorylation of multiple proteins were observed in whole cell lysates prepared from PMN-stimulated epithelial cells. We propose that regulation of PMN transepithelial migration is mediated, in part, by sequential signaling events between migrating PMN and the epithelium.
Skin Graft Rejection Elicited by β2-Microglobulin as a Minor Transplantation Antigen Involves Multiple Effector Pathways: Role of Fas-Fas Ligand Interactions and Th2-Dependent Graft Eosinophil Infiltrates
Authors: Surquin Murielle , Le Moine Alain , Flamand Véronique , Nagy Nathalie , Rombaut Katia , Demoor François-Xavier , Stordeur Patrick , Salmon Isabelle , Guéry Jean-Charles , Goldman Michel , Abramowicz Daniel
Published: April 2014

β2-Microglobulin (β2m)-derived peptides are minor transplantation Ags in mice as β2m-positive skin grafts (β2m+/+) are rejected by genetically β2m-deficient recipient mice (β2m−/−). We studied the effector pathways responsible for the rejection induced by β2-microglobulin-derived minor transplantation Ags. The rejection of β2m+/+ skin grafts by naive β2m−/− mice was dependent on both CD4 and CD8 T cells as shown by administration of depleting mAbs. Experiments performed with β2m−/−CD8−/− double knockout mice grafted with a β2m+/+ MHC class I-deficient skin showed that sensitized CD4 T cells directed at β2m peptides-MHC class II complexes are sufficient to trigger rapid rejection. Rejection of β2m+/+ grafts was associated with the production of IL-5 in vitro, the expression of IL-4 and IL-5 mRNAs in the grafted tissue, and the presence within rejected grafts of a considerable eosinophil infiltrate. Blocking IL-4 and IL-5 in vivo and depleting eosinophils with an anti-CCR3 mAb prevented graft eosinophil infiltration and prolonged β2m+/+ skin graft survival. Lymphocytes from rejecting β2m−/− mice also displayed an increased production of IFN-γ after culture with β2m+/+ minor alloantigens. In vivo neutralization of IFN-γ inhibited skin graft rejection. Finally, β2m+/+ skin grafts harvested from B6lpr/lpr donor mice, which lack a functional Fas molecule, survived longer than wild-type β2m+/+ skin grafts, showing that Fas-Fas ligand interactions are involved in the rejection process. We conclude that IL-4- and IL-5-dependent eosinophilic rejection, IFN-γ-dependent mechanisms, and Fas-Fas ligand interactions are effector pathways in the acute rejection of minor transplantation Ags.
High Immunogenicity of Intracellular Myelin Oligodendrocyte Glycoprotein Epitopes
Authors: Weissert Robert , Kuhle Jens , de Graaf Katrien L. , Wienhold Wolfgang , Herrmann Martin M. , Müller Claudia , Forsthuber Thomas G. , Wiesmüller Karl-Heinz , Melms Arthur
Published: April 2014

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the CNS with associated axonal loss. There is strong evidence for an autoimmune pathogenesis driven by myelin-specific T cells. Myelin oligodendrocyte glycoprotein (MOG) induces a type of experimental autoimmune encephalomyelitis in animals which is very MS-like since there are demyelinating CNS lesions and axonal loss. This underscores the potential role of MOG in MS pathogenesis. We performed a T cell reactivity pattern analysis of MS patients at the onset of relapse or progression of neurological deficits and controls that were stratified for the genetic risk factor HLA-DRB1*1501. For the first time, we show that there is an HLA-DR-restricted promiscuous dominant epitope for CD4+ T cells within the transmembrane/intracellular part of MOG comprising aa 146–154 (FLCLQYRLR). Surprisingly, controls had broader T cell reactivity patterns toward MOG peptides compared with MS patients, and the transmembrane and intracellular parts of MOG were much more immunogenic compared with the extracellular part. Measurements of in vitro binding affinities revealed that HLA-DRB1*1501 molecules bound MOG 146–154 with intermediate and HLA-DRB1*0401 molecules with weak affinities. The binding of MOG 146–154 was comparable or better than myelin basic protein 85–99, which is the dominant myelin basic protein epitope in context with HLA-DRB1*1501 molecules in MS patients. This is the first study in which the data underscore the need to investigate the pathogenic or regulatory role of the transmembrane and intracellular part of MOG for MS in more detail.
Using Tolerance Induced Via the Anterior Chamber of the Eye to Inhibit Th2-Dependent Pulmonary Pathology
Authors: Katagiri Kazumoto , Zhang-Hoover Jie , Mo Jun Song , Stein-Streilein Joan , Streilein J. Wayne
Published: April 2014

Anterior chamber-associated immune deviation (ACAID), a manifestation of ocular immune privilege, prevents Th1-dependent delayed hypersensitivity from developing in response to eye-derived Ags, thereby preserving vision. Since Th2-type cells have recently been shown to mediate destructive inflammation of the cornea, we wondered whether pre-emptive induction of ACAID could inhibit Th2 responses. Using a murine model of OVA -specific, Th2-dependent pulmonary inflammation, we pretreated susceptible mice by injecting OVA alone into the anterior chamber, or by injecting OVA-pulsed, TGF-β2-treated peritoneal exudate cells i.v. These mice were then immunized with OVA plus alum strategy that generates Th2-mediated OVA-specific pulmonary pathology. When pretreated mice were challenged intratracheally with OVA, their bronchoalveolar lavage fluids contained far fewer eosinophils and significantly less IL-4, IL-5, and IL-13 compared with that of positive, nonpretreated controls. Similarly, lung-draining lymph node cells of pretreated mice secreted significantly less IL-4, IL-5, and IL-13 when challenged in vitro with OVA. Moreover, sera from pretreated mice contained much lower titers of OVA-specific IgE Abs. We conclude that Ags injected into the anterior chamber of the eye impair both Th1 and Th2 responses. These results reduce the likelihood that ACAID regulates Th1 responses via a Th2-like mechanism. Thus, immune privilege of the eye regulates inflammation secondary to both Th1- and Th2-type immune responses.
Exogenous Peptides Delivered by Ricin Require Processing by Signal Peptidase for Transporter Associated with Antigen Processing-Independent MHC Class I-Restricted Presentation
Authors: Smith Daniel C. , Gallimore Awen , Jones Emma , Roberts Brenda , Lord J. Michael , Deeks Emma , Cerundolo Vincenzo , Roberts Lynne M.
Published: April 2014

In this study we demonstrate that a disarmed version of the cytotoxin ricin can deliver exogenous CD8+ T cell epitopes into the MHC class I-restricted pathway by a TAP-independent, signal peptidase-dependent pathway. Defined viral peptide epitopes genetically fused to the N terminus of an attenuated ricin A subunit (RTA) that was reassociated with its partner B subunit were able to reach the early secretory pathway of sensitive cells, including TAP-deficient cells. Successful processing and presentation by MHC class I proteins was not dependent on proteasome activity or on recycling of MHC class I proteins, but rather on a functional secretory pathway. Our results demonstrated a role for signal peptidase in the generation of peptide epitopes associated at the amino terminus of RTA. We showed, first, that potential signal peptide cleavage sites located toward the N terminus of RTA can be posttranslationally cleaved by signal peptidase and, second, that mutation of one of these sites led to a loss of peptide presentation. These results identify a novel MHC class I presentation pathway that exploits the ability of toxins to reach the lumen of the endoplasmic reticulum by retrograde transport, and suggest a role for endoplasmic reticulum signal peptidase in the processing and presentation of MHC class I peptides. Because TAP-negative cells can be sensitized for CTL killing following retrograde transport of toxin-linked peptides, application of these results has direct implications for the development of novel vaccination strategies.
Cutting Edge: Tumor Rejection Mediated by NKG2D Receptor-Ligand Interaction Is Dependent upon Perforin
Authors: Hayakawa Yoshihiro , Kelly Janice M. , Westwood Jennifer A. , Darcy Phillip K. , Diefenbach Andreas , Raulet David , Smyth Mark J.
Published: April 2014

We have investigated the primary immunity generated in vivo by MHC class I-deficient and -competent tumor cell lines that expressed the NKG2D ligand retinoic acid early inducible-1 (Rae-1) β. Rae-1β expression on class I-deficient RMA-S lymphoma cells enhanced primary NK cell-mediated tumor rejection in vivo, whereas RMA-Rae-1β tumor cells were rejected by a combination of NK cells and CD8+ T cells. Rae-1β expression stimulated NK cell cytotoxicity and IFN-γ secretion in vitro, but not proliferation. Surprisingly, only NK cell perforin-mediated cytotoxicity, but not production of IFN-γ, was critical for the rejection of Rae-1β-expressing tumor cells in vivo. This distinct requirement for perforin activity contrasts with the NK cell-mediated rejection of MHC class I-deficient RMA-S tumor cells expressing other activating ligands such as CD70 and CD80. Thus, these results indicated that NKG2D acted as a natural cytotoxicity receptor to stimulate perforin-mediated elimination of ligand-expressing tumor cells.
Tumor-Derived Heat Shock Protein 70 Peptide Complexes Are Cross-Presented by Human Dendritic Cells
Authors: Noessner Elfriede , Gastpar Robert , Milani Valeria , Brandl Anna , Hutzler Peter J. S. , Kuppner Maria C. , Roos Miriam , Kremmer Elisabeth , Asea Alexzander , Calderwood Stuart K. , Issels Rolf D.
Published: April 2014

Our study demonstrates that tumor-derived heat shock protein (HSP)70 chaperones a tyrosinase peptide and mediates its transfer to human immature dendritic cells (DCs) by receptor-dependent uptake. Human tumor-derived HSP70 peptide complexes (HSP70-PC) thus have the immunogenic potential to instruct DCs to cross-present endogenously expressed, nonmutated, and tumor antigenic peptides that are shared among tumors of the melanocytic lineage for T cell recognition. T cell stimulation by HSP70-instructed DCs is dependent on the Ag bound to HSP70 in that only DCs incubated with HSP70-PC purified from tyrosinase-positive (HSP70-PC/tyr+) but not from tyrosinase-negative (HSP70-PC/tyr−) melanoma cells resulted in the specific activation of the HLA-A*0201-restricted tyrosinase peptide-specific cytotoxic T cell clone. HSP70-PC-mediated T cell stimulation is very efficient, delivering the tyrosinase peptide at concentrations as low as 30 ng/ml of HSP70-PC for T cell recognition. Receptor-dependent binding of HSP70-PC and active cell metabolism are prerequisites for MHC class I-restricted cross-presentation and T cell stimulation. T cell stimulation does not require external DC maturation signals (e.g., exogenously added TNF-α), suggesting that signaling DC maturation is an intrinsic property of the HSP70-PC itself and related to receptor-mediated binding. The cross-presentation of a shared human tumor Ag together with the exquisite efficacy are important new aspects for HSP70-based immunotherapy in clinical anti-cancer vaccination strategies, and suggest a potential extension of HSP70-based vaccination protocols from a patient-individual treatment modality to its use in an allogeneic setting.