bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms of Soluble Cytokine Receptor Generation
Authors: Levine Stewart J.
Published: April 2014

Soluble cytokine receptors regulate inflammatory and immune events by functioning as agonists or antagonists of cytokine signaling. As such, they act within complex receptor systems that include signaling receptors, nonsignaling decoy receptors, receptor-associated proteins, and soluble receptor antagonists. Soluble cytokine receptors can be generated by several mechanisms, which include proteolytic cleavage of receptor ectodomains, alternative splicing of mRNA transcripts, transcription of distinct genes that encode soluble cytokine-binding proteins, release of full-length receptors within the context of exosome-like vesicles, and cleavage of GPI-anchored receptors. Furthermore, the important role of soluble cytokine receptors in regulating host defense mechanisms is evidenced by viruses that encode soluble homologues of mammalian receptors and thereby evade innate host immune responses via the sequestration of essential cytokines.
Deranged Early T Cell Development in Immunodeficient Strains of Nonobese Diabetic Mice
Authors: Yui Mary A. , Rothenberg Ellen V.
Published: April 2014

NOD mice exhibit defects in T cell functions that have been postulated to contribute to diabetes susceptibility in this strain. However, early T cell development in NOD mice has been largely unexplored. NOD mice with the scid mutation and Rag1 deficiency were analyzed for pre-T cell development in the NOD genetic background. These strains reveal an age-dependent, programmed breakdown in β selection checkpoint enforcement. At 5–8 wk of age, even in the absence of TCRβ expression, CD4+ and CD4+CD8+ blasts appear spontaneously. However, these breakthrough cells fail to restore normal thymic cellularity. The breakthrough phenotype is recessive in hybrid (NOD×B6)F1-scid and -Rag1null mice. The breakthrough cells show a mosaic phenotype with respect to components of the β selection program. They mimic normal β selection by up-regulating germline TCR-Cα transcripts, CD2, and Bcl-xL and down-regulating Bcl-2. However, they fail to down-regulate transcription factors HEB-alt and Hes1 and initially express aberrantly high levels of Spi-B, c-kit (CD117), and IL-7Rα. Other genes examined distinguish this form of breakthrough from previously reported models. Some of the abnormalities appear first in a cohort of postnatal thymocytes as early as the double-negative 2/double-negative 3 transitional stage. Thus, our results reveal an NOD genetic defect in T cell developmental programming and checkpoint control that permits a subset of the normal outcomes of pre-TCR signaling to proceed even in the absence of TCRβ rearrangement. Furthermore, this breakthrough may initiate thymic lymphomagenesis that occurs with high frequency in both NOD-scid and -Rag1null mice.
Rapid Induction of Splenic and Peritoneal B-1a Cells in Adult Mice by Thymus-Independent Type-2 Antigen
Authors: Whitmore Alan C. , Neely Harold R. , Diz Ramiro , Flood Patrick M.
Published: April 2014

We have produced a transgenic mouse (PV1TgL) that can only generate B lymphocytes with an Ig receptor specific for the synthetic polymer polyvinyl pyrrolidinone. Before immunization, bone marrow B cell numbers are very low, and peripheral lymphoid organs are almost devoid of B cells, confirming the role of positive selection by Ag in the development of mature B cell populations. The predominant population of B cells in the spleens of naive adult PV1TgL mice have most of the characteristics of marginal zone B cells, including anatomical location in the peripheral areas of the splenic white pulp. After immunization, a new population of B cells appears in the spleen with the characteristics of B-1 cells. Similar cells also appear somewhat later in the peritoneal cavity. Our findings suggest that immunization with a thymus-independent Ag can lead to the appearance and expansion of Ag-reactive B-1 cells in an adult mouse.
The Selective Increase in Caspase-3 Expression in Effector but Not Memory T Cells Allows Susceptibility to Apoptosis
Authors: Sabbagh Laurent , Kaech Susan M. , Bourbonnière Martin , Woo Minna , Cohen Luchino Y. , Haddad Elias K. , Labrecque Nathalie , Ahmed Rafi , Sékaly Rafick-Pierre
Published: April 2014

Caspases play a central role in T lymphocyte activation and death. We have demonstrated previously that caspase-3, an effector molecule for activation-induced cell death (AICD), is processed following T cell activation in the absence of apoptosis. We report in this study that caspase-3 mRNA levels were selectively increased in peripheral T cells, following Ag receptor-mediated activation. The up-regulation of caspase-3 mRNA was confined to cells in the early phases of the cell cycle (G0/G1) and was independent of IL-2 signaling. This increase led to the renewal of procaspase-3 as evidenced by a 6-fold up-regulation of the zymogen in nonapoptotic stimulated T cells. The increase of mRNA levels and of both the zymogen and the cleaved forms of caspase-3 was observed in in vivo stimulated Ag-specific effector, but not memory T cells, correlating with the enhanced susceptibility of effector T cells to AICD. Furthermore, we confirm that caspase-3 levels directly influence the sensitivity of activated T cells to apoptosis, as shown using T lymphocytes isolated from caspase-3 heterozygous and knockout mice. These findings indicate that the selective up-regulation of caspase-3 transcription is required to maintain the cytoplasmic levels of this protease, which control AICD and T cell homeostasis.
Functionally Impaired HIV-Specific CD8 T Cells Show High Affinity TCR-Ligand Interactions
Authors: Ueno Takamasa , Tomiyama Hiroko , Fujiwara Mamoru , Oka Shinichi , Takiguchi Masafumi
Published: April 2014

We eventually isolated two different clonotypic CD8 T cell subsets recognizing an HIV Pol-derived epitope peptide (IPLTEEAEL) in association with HLA-B35 from a chronic HIV-infected patient. By kinetic analysis experiments, the subsets showed a >3-fold difference in half-lives for the HLA tetramer in complex with the Pol peptide. In functional assays in vitro and ex vivo, both subsets showed substantial functional avidity toward peptide-loaded cells. However, the high affinity subset did not show cytolytic activity, cytokine production, or proliferation activity toward HIV-infected cells, whereas the moderate affinity one showed potent activities. Furthermore, using ectopic expression of each of the TCR genes into primary human CD8 T cells, the CD8 T cells transduced with the high affinity TCR showed greater binding activity toward the tetramer and impaired cytotoxic activity toward HIV-infected cells, corroborating the results obtained with parental CD8 T cells. Taken together, these data indicate that impaired responsiveness of T cells toward HIV-infected cells can occur at the level of TCR-ligand interactions, providing us further insight into the immune evasion mechanisms by HIV.
Prostaglandin E2-Dependent Enhancement of Tissue Inhibitors of Metalloproteinases-1 Production Limits Dendritic Cell Migration through Extracellular Matrix
Authors: Baratelli Felicita E. , Heuzé-Vourc’h Nathalie , Krysan Kostyantyn , Dohadwala Mariam , Riedl Karen , Sharma Sherven , Dubinett Steven M.
Published: April 2014

Dendritic cell (DC) migration is crucial for the initiation of immune responses. The balance between metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) has been shown to modulate DC migration. PGE2, which is overproduced in a wide variety of human malignancies, has been implicated in MMP and TIMP regulation in various cells, including monocytes. In the present study, we hypothesized that tumor-derived PGE2 would affect DC migratory capacity through the extracellular matrix (ECM) by altering MMP and TIMP balance. Treatment of monocyte-derived immature DC with exogenous PGE2 induced TIMP-1 secretion but not MMP-9 production and was correlated with reduced DC migration through ECM. Because recombinant TIMP-1 replicated PGE2 inhibition of DC migration while anti-TIMP-1 neutralizing Ab reversed it, we conclude that PGE2-mediated induction of TIMP-1 was responsible for the reduced migration of PGE2-treated DC. Similarly, DC cultured for 48 h in supernatants from cyclooxygenase-2 overexpressing lung cancer cells that secrete high levels of PGE2, exhibited decreased migration through ECM. Finally, analysis of E prostanoid receptor expression and their selective inhibition revealed that the enhanced TIMP-1 secretion in PGE2-treated DC was mediated predominantly by the E prostanoid receptor 2. These findings indicate that PGE2-dependent enhancement of TIMP-1 production causes reduced migration of DC through ECM.
Target Antigens Determine Graft-versus-Host Disease Phenotype
Authors: Kaplan Daniel H. , Anderson Britt E. , McNiff Jennifer M. , Jain Dhanpat , Shlomchik Mark J. , Shlomchik Warren D.
Published: April 2014

Chronic graft-vs-host disease (cGVHD) is an increasingly frequent complication of allogeneic stem cell transplantation. Phenotypically, cGVHD differs from patient to patient; in particular, a subset of patients develops extensive cutaneous fibrosis. Similarly, graft-vs-host disease (GVHD) is distinct in inbred murine donor:recipient pairings, indicating a genetic component to disease phenotype. The B10.D2 → BALB/c (H-2d) strain pairing uniquely recapitulates key pathologic features of fibrotic human cutaneous cGVHD. To distinguish whether this genetic component is due to differences in genes that modulate immune responses or to the specific Ags targeted, we asked whether skin-dominant cGVHD also develops in the B10 → BALB.B (H-2b) and B10.BR → BALB.K (H-2k) MHC-congenic pairings. Because each MHC haplotype presents different peptides and selects different T cell repertoires, GVHD in each donor:recipient pair undoubtedly targets different Ags. We found that, in contrast to BALB/c recipients, BALB.B mice never manifested skin disease while BALB.K mice developed a modified form of skin disease. Instead, BALB.B and BALB.K recipients developed systemic GVHD which was absent in BALB/c mice. Moreover, in (B10 × B10.D2)F1 → (BALB.B × BALB/c)F1 H-2b/d transplants, recipients developed both cutaneous and systemic disease. Thus, the selection of immunodominant Ags determines the target and character of GVHD, providing insight into the genetic basis for different forms of GVHD.
Evolution of Transcriptional Control of theIgHLocus: Characterization, Expression, and Function of TF12/HEB Homologs of the Catfish
Authors: Hikima Jun-ichi , Cioffi Christopher C. , Middleton Darlene L. , Wilson Melanie R. , Miller Norman W. , Clem L. William , Warr Gregory W.
Published: April 2014

The transcriptional enhancer (Eμ3′) of the IgH locus of the channel catfish, Ictalurus punctatus, differs from enhancers of the mammalian IgH locus in terms of its position, structure, and function. Transcription factors binding to multiple octamer motifs and a single μE5 motif (an E-box site, consensus CANNTG) interact for its function. E-box binding transcription factors of the class I basic helix-loop-helix family were cloned from a catfish B cell cDNA library in this study, and homologs of TF12/HEB were identified as the most highly represented E-proteins. Two alternatively spliced forms of catfish TF12 (termed CFEB1 and -2) were identified and contained regions homologous to the basic helix-loop-helix and activation domains of other vertebrate E-proteins. CFEB message is widely expressed, with CFEB1 message predominating over that of CFEB2. Both CFEB1 and -2 strongly activated transcription from a μE5-dependent artificial promoter. In catfish B cells, CFEB1 and -2 also activated transcription from the core region of the catfish IgH enhancer (Eμ3′) in a manner dependent on the presence of the μE5 site. Both CFEB1 and -2 bound the μE5 motif, and formed both homo- and heterodimers. CFEB1 and -2 were weakly active or inactive (in a promoter-dependent fashion) in mammalian B-lineage cells. Although E-proteins have been highly conserved in vertebrate evolution, the present results indicate that, at the phylogenetic level of a teleost fish, the TF12/HEB homolog differs from that of mammals in terms of 1) its high level of expression and 2) the presence of isoforms generated by alternative RNA processing.
CD4 Phosphorylation Partially Reverses Nef Down-Regulation of CD4
Authors: Jin Yong-Jiu , Zhang Xiaoping , Boursiquot J. Gildade , Burakoff Steven J.
Published: April 2014

HIV Nef down-regulates CD4 from the cell surface in the absence of CD4 phosphorylation, whereas PMA down-regulates CD4 through a phosphorylation-dependent pathway. In this study we show that the down-regulation of CD4 in human Jurkat T cells expressing Nef was nearly complete (∼95%), whereas that induced by PMA was partial (∼40%). Unexpectedly, treating T cells expressing Nef with PMA restored the surface CD4 up to 35% of the steady state level. Both mutating the phosphorylation sites in the CD4 cytoplasmic tail (Ser408 and Ser415) and the use of a protein kinase C inhibitor, bisindolylmaleimide1, abolished the restoration of surface CD4, suggesting that the restoration required CD4 phosphorylation. CD4 and Nef could be cross-linked by a chemical cross-linker, 3,3-dithiobis[sulfosuccinimidyl-propionate], in control T cell membranes, but not in PMA-treated T cell membrane, suggesting that CD4 and Nef interacted with each other in T cells, and the phosphorylation disrupted the CD4-Nef interaction. We propose that this dissociation switches CD4 internalization from the Nef-mediated, nearly complete down-regulation to a phosphorylation-dependent, partial down-regulation, resulting in a net gain of CD4 on the T cell surface.
Altered Th1 Cell Differentiation Programming by CIITA Deficiency
Authors: Patel Dipak R. , Kaplan Mark H. , Chang Cheong-Hee
Published: April 2014

CD4 T cell differentiation is a complex process affected by many transcription factors interacting in a tightly regulated manner. We have previously shown that CIITA-deficient mouse Th1 cells expressed Th2-type cytokines, while IFN-γ expression was normal. In this study, we show that CIITA-deficient Th1 cells contain three distinct populations: cells secreting IL-4 alone, IFN-γ alone, and both IL-4 and IFN-γ together. This novel phenotype is stable over multiple rounds of stimulation in the presence of Th1-inducing factors. CIITA-deficient Th1 cells require TCR-mediated signaling to express Th2 cytokines, and this occurs with similar kinetics as wild-type Th2 cells. Both GATA-3 and IL-4 appear to be required for CIITA-deficient Th1 cells to express Th2-type cytokines. Interestingly, however, CIITA-deficient Th1 cells can produce IL-4 in the absence of exogenous IL-4. Introducing either CIITA or antisense GATA-3 during Th1 differentiation partially reduces Th2-type cytokine expression. With the exception of Th2-type cytokine expression, Th1 differentiation occurs normally in the absence of CIITA, as measured by expression of T-bet, IL-12Rβ2, IL-18Rα, and IFN-γ. Therefore, CIITA plays a key role to repress Th2-type cytokine expression as naive CD4 T cells differentiate toward the Th1 lineage.
B Lymphocyte-Induced Maturation Protein (Blimp)-1, IFN Regulatory Factor (IRF)-1, and IRF-2 Can Bind to the Same Regulatory Sites
Authors: Kuo Tracy C. , Calame Kathryn L.
Published: April 2014

The transcriptional repressor B lymphocyte-induced maturation protein-1 (Blimp-1) is expressed in some differentiated cells and is required for terminal differentiation of B cells. To facilitate identification of Blimp-1 target genes, we have determined the optimal DNA recognition sequence for Blimp-1. The consensus is very similar to a subset of sites recognized by IFN regulatory factors (IRFs) that contain the sequence GAAAG. By binding competition and determination of equilibrium dissociation constants, we show that Blimp-1, IRF-1, and IRF-2 have similar binding affinities for functionally important regulatory sites containing this sequence. However, Blimp-1 does not bind to all IRF sites, and specifically does not recognize IRF-4/PU.1 or IRF-8 sites lacking the GAAAG sequence. Chromatin immunoprecipitation studies showed that Blimp-1, IRF-1, and IRF-2 all bind the IFN-β promoter in vivo, as predicted by the in vitro binding parameters, and in cotransfections Blimp-1 inhibits IRF-1-dependent activation of the IFN-β promoter. Thus, our data suggest that Blimp-1 competes in vivo with a subset of IRF proteins and help predict the sites and IRF family members that may be affected.
The Interaction between GATA Proteins and Activator Protein-1 Promotes the Transcription ofIL-13in Mast Cells
Authors: Masuda Akio , Yoshikai Yasunobu , Kume Hiroaki , Matsuguchi Tetsuya
Published: April 2014

IL-13 is considered to be a key modulator in the pathogenesis of Th2-induced allergic inflammation, although little is known about the regulation of IL-13 transcription in mast cells. In T cells, involvement of GATA-3 in cell type-specific expression of the IL-13 gene has been reported. However, the mechanisms that induce rapid transactivation of the IL-13 gene in response to various types of stimulation have hitherto remained unknown. In this report, we describe our investigation of the promoter region necessary for IL-13 transcription; we have found that both AP-1 and GATA proteins are indispensable for IL-13 transcription in mouse mast cells. In our investigation, we focused on the functional interaction between GATA and AP-1 in the IL-13 promoter context. Transfection experiments have revealed that GATA-1 and GATA-2 proteins are able to associate with AP-1 proteins. We have also shown that overexpression of GATA-1 induced excess AP-1 binding to the IL-13 promoter as well as a significant increase in IL-13 production in mast cells. The results of the present study have shown that direct interaction between AP-1 and GATA proteins plays an important role in IL-13 transcription in mast cells.
Shark Ig Light Chain Junctions Are as Diverse as in Heavy Chains
Authors: Fleurant Marshall , Changchien Lily , Chen Chin-Tung , Flajnik Martin F. , Hsu Ellen
Published: April 2014

We have characterized a small family of four genes encoding one of the three nurse shark Ig L chain isotypes, called NS5. All NS5 cDNA sequences are encoded by three loci, of which two are organized as conventional clusters, each consisting of a V and J gene segment that can recombine and one C region exon; the third contains a germline-joined VJ in-frame and the fourth locus is a pseudogene. This is the second nurse shark L chain type where both germline-joined and split V-J organizations have been found. Since there are only two rearranging Ig loci, it was possible for the first time to examine junctional diversity in defined fish Ig genes, comparing productive vs nonproductive rearrangements. N region addition was found to be considerably more extensive in length and in frequency than any other vertebrate L chain so far reported and rivals that in H chain. We put forth the speculation that the unprecedented efficiency of N region addition (87–93% of NS5 sequences) may be a result not only of simultaneous H and L chain rearrangement in the shark but also of processing events that afford greater accessibility of the V or J gene coding ends to terminal deoxynucleotidyltransferase.
Exclusion and Inclusion of TCRα Proteins during T Cell Development in TCR-Transgenic and Normal Mice
Authors: Lacorazza H. Daniel , Nikolich-Žugich Janko
Published: April 2014

Allelic exclusion of immune receptor genes (and molecules) is incompletely understood. With regard to TCRαβ lineage T cells, exclusion at the tcr-b, but not tcr-a, locus seems to be strictly controlled at the locus rearrangement level. Consequently, while nearly all developing TCRαβ thymocytes express a single TCRβ protein, many thymocytes rearrange and express two different TCRα chains and, thus, display two αβTCRs on the cell surface. Of interest, the number of such dual TCR-expressing cells is appreciably lower among the mature T cells. To understand the details of TCR chain regulation at various stages of T cell development, we analyzed TCR expression in mice transgenic for two rearranged αβTCR. We discovered that in such TCR double-transgenic (TCRdTg) mice peripheral T cells were functionally monospecific. Molecularly, this monospecificity was due to TCRα exclusion: one transgenic TCRα protein was selectively down-regulated from the thymocyte and T cell surface. In searching for the mechanism(s) governing this selective TCRα down-regulation, we present evidence for the role of protein tyrosine kinase signaling and coreceptor involvement. This mechanism may be operating in normal thymocytes.
Conformational Restraints and Flexibility of 14-Meric Peptides in Complex with HLA-B*3501
Authors: Probst-Kepper Michael , Hecht Hans-Jürgen , Herrmann Hanne , Janke Viktoria , Ocklenburg Frank , Klempnauer Jürgen , van den Eynde Benoit J. , Weiss Siegfried
Published: April 2014

Human HLA-B*3501 binds an antigenic peptide of 14-aa length derived from an alternative reading frame of M-CSF with high affinity. Due to its extraordinary length, the exact HLA binding mode was unpredictable. The crystal structure of HLA-B*3501 at 1.5 Å shows that the N and C termini of the peptide are embedded in the A and F pockets, respectively, similar to a peptide of normal length. The central part of the 14-meric peptide bulges flexibly out of the groove. Two variants of the alternative reading frame of M-CSF peptide substituted at P2 or P2 and P9 with Ala display weak or no T cell activation. Their structure differs mainly in flexibility and conformation from the agonistic peptide. Moreover, the variants induce subtle changes of MHC α-helical regions implicated as critical for TCR contact. The TCR specifically recognizing this peptide/MHC complex exhibits CDR3 length within the normal range, suggesting major conformational adaptations of this receptor upon peptide/MHC binding. Thus, the potential antigenic repertoire recognizable by CTLs is larger than currently thought.
The IL-27 Receptor (WSX-1) Is an Inhibitor of Innate and Adaptive Elements of Type 2 Immunity
Authors: Artis David , Villarino Alejandro , Silverman Michael , He Weimian , Thornton Elizabeth M. , Mu Sharon , Summer Shamin , Covey Todd M. , Huang Elaine , Yoshida Hiroki , Koretzky Gary , Goldschmidt Michael , Wu Gary D. , de Sauvage Fred , Miller Hugh R. P. , Saris Christiaan J. M. , Scott Phillip , Hunter Christopher A.
Published: April 2014

Although previous studies have investigated the role of IL-27/WSX-1 interactions in the regulation of Th1 responses, little is known about their role in regulating Th2-type responses. Studies presented in this work identify a direct role for IL-27/WSX-1 interactions in the negative regulation of type 2 responses independent of effects on type 1 cytokines. WSX-1−/− mice infected with the gastrointestinal helminth Trichuris muris displayed accelerated expulsion of parasites and the development of exaggerated goblet cell hyperplasia and mastocytosis in the gut due to increased production of Th2 cytokines. Enhanced mast cell activity in the absence of WSX-1 was consistent with the ability of wild-type mast cells to express this receptor. In addition, IL-27 directly suppressed CD4+ T cell proliferation and Th2 cytokine production. Together, these studies identify a novel role for IL-27/WSX-1 in limiting innate and adaptive components of type 2 immunity at mucosal sites.
Cross-Presentation of Listeria-Derived CD8 T Cell Epitopes Requires Unstable Bacterial Translation Products
Authors: Janda Jozef , Schöneberger Petra , Škoberne Mojca , Messerle Martin , Rüssmann Holger , Geginat Gernot
Published: April 2014

Presentation of bacteria-derived CD8 T cell epitopes by dendritic cells (DC) requires either their direct infection or that DC acquire and cross-present Ags from other infected cells. We found that cross-presentation of Listeria monocytogenes-derived CD8 T cell epitopes was much stronger than direct Ag presentation by infected murine DC. Cross-presentation of Listeria-derived CD8 T cell epitopes showed unique physiological requirements. It was dependent upon the delivery of unstable bacterial translation products by infected, but still viable, Ag donor cells. Cross-presentation was enhanced both when unstable translation products in infected Ag donor cells were protected from proteasomal degradation and when the production of misfolded bacterial proteins was increased. The requirement of unstable translation products for cross-presentation may represent a novel pathway that functions to focus the CD8 T cell response toward epitopes derived from newly synthesized proteins.
Pseudomonas aeruginosa Flagellin and Alginate Elicit Very Distinct Gene Expression Patterns in Airway Epithelial Cells: Implications for Cystic Fibrosis Disease
Authors: Cobb Laura M. , Mychaleckyj Josyf C. , Wozniak Daniel J. , López-Boado Yolanda S.
Published: April 2014

Infection with the opportunistic pathogen Pseudomonas aeruginosa remains a major health concern. Two P. aeruginosa phenotypes relevant in human disease include motility and mucoidy. Motility is characterized by the presence of flagella and is essential in the establishment of acute infections, while mucoidy, defined by the production of the exopolysaccharide alginate, is critical in the development of chronic infections, such as the infections seen in cystic fibrosis patients. Indeed, chronic infection of the lung by mucoid P. aeruginosa is a major cause of morbidity and mortality in cystic fibrosis patients. We have used Calu-3 human airway epithelial cells to investigate global responses to infection with motile and mucoid P. aeruginosa. The response of airway epithelial cells to exposure to P. aeruginosa motile strains is characterized by a specific increase in gene expression in pathways controlling inflammation and host defense. By contrast, the response of airway epithelia to the stimuli presented by mucoid P. aeruginosa is not proinflammatory and, hence, may not be conducive to the effective elimination of the pathogen. The pattern of gene expression directed by flagellin, but not alginate, includes innate host defense genes, proinflammatory cytokines, and chemokines. By contrast, infection with alginate-producing P. aeruginosa results in an overall attenuation of host responses and an antiapoptotic effect.
Human Monoclonal Antibodies toPseudomonas aeruginosaAlginate That Protect against Infection by Both Mucoid and Nonmucoid Strains
Authors: Pier Gerald B. , Boyer Debra , Preston Michael , Coleman Fadie T. , Llosa Nicolas , Mueschenborn-Koglin Simone , Theilacker Christian , Goldenberg Hannah , Uchin Jeffrey , Priebe Gregory P. , Grout Martha , Posner Marshall , Cavacini Lisa
Published: April 2014

Two fully human mAbs specific for epitopes dependent on intact carboxylate groups on the C6 carbon of the mannuronic acid components of Pseudomonas aeruginosa alginate were found to promote phagocytic killing of both mucoid and nonmucoid strains as well as protection against both types of strains in a mouse model of acute pneumonia. The specificity of the mAbs for alginate was determined by ELISA and killing assays. Some strains of P. aeruginosa did not make detectable alginate in vitro, but in vivo protection against lethal pneumonia was obtained and shown to be due to rapid induction of expression of alginate in the murine lung. No protection against strains genetically unable to make alginate was achieved. These mAbs have potential to be passive therapeutic reagents for all strains of P. aeruginosa and the results document that alginate is a target for the proper type of protective Ab even when expressed at low levels on phenotypically nonmucoid strains.
Urokinase Induces Basophil Chemotaxis through a Urokinase Receptor Epitope That Is an Endogenous Ligand for Formyl Peptide Receptor-Like 1 and -Like 2
Authors: de Paulis Amato , Montuori Nunzia , Prevete Nella , Fiorentino Isabella , Rossi Francesca Wanda , Visconte Valeria , Rossi Guido , Marone Gianni , Ragno Pia
Published: April 2014

Basophils circulate in the blood and are able to migrate into tissues at sites of inflammation. Urokinase plasminogen activator (uPA) binds a specific high affinity surface receptor (uPAR). The uPA-uPAR system is crucial for cell adhesion and migration, and tissue repair. We have investigated the presence and function of the uPA-uPAR system in human basophils. The expression of uPAR was found at both mRNA and protein levels. The receptor was expressed on the cell surface of basophils, in the intact and cleaved forms. Basophils did not express uPA at either the protein or mRNA level. uPA (10−12–10−9 M) and its uPAR-binding N-terminal fragment (ATF) were potent chemoattractants for basophils, but did not induce histamine or cytokine release. Inactivation of uPA enzymatic activity by di-isopropyl fluorophosphate did not affect its chemotactic activity. A polyclonal Ab against uPAR inhibited uPA-dependent basophil chemotaxis. The uPAR-derived peptide 84–95 (uPAR84–95) induced basophil chemotaxis. Basophils expressed mRNA for the formyl peptide receptors formyl peptide receptor (FPR), FPR-like 1 (FPRL1), and FPRL2. The FPR antagonist cyclosporin H prevented chemotaxis induced by FMLP, but not that induced by uPA and uPAR84–95. Incubation of basophils with low and high concentrations of FMLP, which desensitize FPR and FPRL1, respectively, but not FPRL2, slightly reduced the chemotactic response to uPA and uPAR84–95. In contrast, desensitization with WKYMVm, which also binds FPRL2, markedly inhibited the response to both molecules. Thus, uPA is a potent chemoattractant for basophils that seems to act through exposure of the chemotactic uPAR epitope uPAR84–95, which is an endogenous ligand for FPRL2 and FPRL1.
A Role for NF-κB Subunits p50 and p65 in the Inhibition of Lipopolysaccharide-Induced Shock
Authors: Gadjeva Mihaela , Tomczak Michal F. , Zhang Ming , Wang Yan Yan , Dull Karen , Rogers Arlin B. , Erdman Susan E. , Fox James G. , Carroll Michael , Horwitz Bruce H.
Published: April 2014

To evaluate the possibility that NF-κB subunits p50 and p65 have a role in limiting the systemic inflammatory response induced by endotoxin, we compared the susceptibility of wild-type (WT), p65+/−, p50−/−, and p50−/−p65+/− (3X) mice to LPS-induced shock. Interestingly, whereas p65+/− mice were no more sensitive than WT mice to LPS-induced shock, 3X mice were exquisitely sensitive to the toxic effects of LPS. Mice lacking p50 alone displayed an intermediate phenotype. Sensitivity to LPS was a property of the innate immune system and was characterized by elevated circulating levels of TNF in both p50−/− and 3X mice. The ability of LPS to induce shock depended upon TNF, and 3X mice were significantly more sensitive to the toxic effects of TNF than were p50-deficient mice. The expression of several LPS-inducible proinflammatory genes, including IFN-γ, was significantly higher within the spleens of p50−/− mice than in the spleens of WT mice, and interestingly, the expression of IFN-γ was augmented still further within the spleens of 3X mice. These results demonstrate that NF-κB subunits p50 and p65 have critical inhibitory functions during the systemic response to LPS and raise the possibility that these functions could be essential in preventing mortality associated with systemic inflammatory response syndromes.
Long-Term Stable Expanded Human CD4+ T Cell Clones Specific for Human Cytomegalovirus Are Distributed in Both CD45RAhigh and CD45ROhigh Populations
Authors: Weekes Michael P. , Wills Mark R. , Sissons J. G. Patrick , Carmichael Andrew J.
Published: April 2014

T cells play an important role in the control of human CMV (HCMV) infection. Peripheral blood CD4+ T cell proliferative responses to the HCMV lower tegument protein pp65 have been detected in most healthy HCMV carriers. To analyze the clonal composition of the CD4+ T cell response against HCMV pp65, we characterized three MHC class II-restricted peptide epitopes within pp65 in virus carriers. In limiting dilution analysis, we observed high frequencies of pp65 peptide-specific CD4+ T cells, many of which expressed peptide-specific cytotoxicity in addition to IFN-γ secretion. We analyzed the clonal composition of CD4+ T cells specific for defined HCMV peptides by generating multiple independent peptide-specific CD4+ clones and sequencing the TCR β-chain. In a given carrier, most of the CD4+ clones specific for a defined pp65 peptide had identical TCR nucleotide sequences. We used clonotype oligonucleotide probing to quantify the size of individual peptide-specific CD4+ clones in whole PBMC and in purified subpopulations of CD45RAhighCD45ROlow and CD45RAlowCD45ROhigh cells. Individual CD4+ T cell clones could be large (0.3–1.5% of all CD4+ T cells in PBMC) and were stable over time. Cells of a single clone were distributed in both the CD45RAhigh and CD45ROhigh subpopulations. In one carrier, the virus-specific clone was especially abundant in the small CD28−CD45RAhigh CD4+ T cell subpopulation. Our study demonstrates marked clonal expansion and phenotypic heterogeneity within daughter cells of a single virus-specific CD4+ T cell clone, which resembles that seen in the CD8+ T cell response against HCMV pp65.
Identification of a Human HLA-E-Restricted CD8+ T Cell Subset in Volunteers Immunized withSalmonella entericaSerovar Typhi Strain Ty21a Typhoid Vaccine
Authors: Salerno-Gonçalves Rosângela , Fernandez-Viña Marcelo , Lewinsohn David M. , Sztein Marcelo B.
Published: April 2014

Our previous studies in volunteers immunized with Salmonella enterica serovar Typhi (S. Typhi) have suggested an important role for CD8+ T cells in host defense. In this study we describe a novel subset of nonclassical human HLA-E-restricted S. Typhi-specific CD8+ T cells derived from PBMC of Ty21a typhoid vaccinees. CD3+CD8+CD4−CD56− T cells effectively killed S. Typhi-infected targets regardless of whether they share classical HLA class I molecules with them, by a FAS-independent, granule-dependent mechanism, as evidenced by induction of granzyme B release and the blocking effects of concanamycin and strontium ions. The expression of HLA-E Ags, but not CD1-a, -b, or -c, on the membrane of S. Typhi-infected targets rendered them susceptible to lysis. Moreover, anti-HLA-E Abs partially blocked these responses. We also demonstrated that presentation of S. Typhi Ags via HLA-E could stimulate IFN-γ production. Increases in the net frequency of IFN-γ spot-forming cells were observed in the presence of targets coated with peptides that contain S. Typhi GroEL HLA-E binding motifs. These results demonstrate that HLA-E binds nonamer peptides derived from bacterial proteins and trigger CD8+-mediated lysis and IFN-γ production when exposed to infected targets, raising the possibility that this novel effector mechanism might contribute to host defense against intracellular bacterial infections.
Cellular Immune Responses to the Hepatitis B Virus Polymerase
Authors: Mizukoshi Eishiro , Sidney John , Livingston Brian , Ghany Marc , Hoofnagle Jay H. , Sette Alessandro , Rehermann Barbara
Published: April 2014

CD4+ T cells play an important role in hepatitis B virus (HBV) infection by secretion of Th1 cytokines that down-regulate HBV replication, and by promoting CD8+ T cell and B cell responses. We have identified and characterized 10 CD4+ T cell epitopes within polymerase and used them to analyze the immunological effects of long-term antiviral therapy as compared with spontaneous recovery from HBV infection. Candidate epitopes were tested for binding to 14 HLA-DR molecules and in IFN-γ ELISPOT and cytotoxicity assays using peripheral blood lymphocytes from 66 HBV-infected patients and 16 uninfected controls. All 10 epitopes bound with high affinity to the most prevalent HLA-DR Ags, were conserved among HBV genomes, and induced IFN-γ responses from HBV-specific CD4+ T cells. Several epitopes contained nested MHC class I motifs and stimulated HBV-specific IFN-γ production and cytotoxicity of CD8+ T cells. HBV polymerase-specific responses were more frequent during acute, self-limited hepatitis and after recovery (12 of 18; 67%) than during chronic hepatitis (16 of 48 (33%); p = 0.02). Antiviral therapy of chronic patients restored HBV polymerase and core-specific T cell responses during the first year of treatment, but thereafter, responses decreased and, after 3 years, were no more frequent than in untreated patients. Decreased T cell responsiveness during prolonged therapy was associated with increased prevalence of lamivudine-resistant HBV mutants and increased HBV titers. The data provide a rationale for the combination of antiviral and immunostimulatory therapy. These newly described HBV polymerase epitopes could be a valuable component of a therapeutic vaccine for a large and ethnically diverse patient population.
Hypoallergenic Variants of the Major Latex Allergen Hev b 6.01 Retaining Human T Lymphocyte Reactivity
Authors: Drew Alexander C. , Eusebius Nirupama P. , Kenins Linda , de Silva Harini D. , Suphioglu Cenk , Rolland Jennifer M. , O’Hehir Robyn E.
Published: April 2014

Hev b 6.01 is a major allergen of natural rubber latex with sensitization of 70–86% of latex glove-allergic subjects. Recently, we mapped the immunodominant T cell sites of Hev b 6.01 to the highly IgE-reactive hevein (Hev b 6.02) domain. Hev b 6.01 contains 14 cysteine residues with multiple disulphide bridges stabilizing tertiary conformation. With the goal of a standardized specific immunotherapy we developed hypoallergenic Hev b 6.01 mutants by site-directed mutagenesis of selected cysteine residues (3, 12, 17, and 41) within the Hev b 6.02 domain. Peptides corresponding to the Hev b 6.02 domain of two of the mutants were also synthesized. These mutants and peptide variants showed markedly decreased or ablated latex-allergic patient serum IgE binding by immunoblotting and ELISA. Basophil activation testing confirmed markedly decreased activation with successive cysteine substitutions of the mutants and complete abrogation with the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide. Retention of T cell reactivity is crucial for effective specific immunotherapy and all mutants and peptide variants maintained their latex-specific T cell reactivity. The ablated allergenicity but retained T cell reactivity of the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide suggests this peptide is a suitable candidate for inclusion in a latex immunotherapy preparation.
Costimulation Requirements of Induced Murine Systemic Autoimmune Disease
Authors: Pollard K. Michael , Arnush Marc , Hultman Per , Kono Dwight H.
Published: April 2014

Costimulation between T cells and APC is required for productive immune responses. A number of receptor/ligand pairs have been shown to mediate costimulation, including CD28/B7 molecules (CD80 and CD86), CD40/CD40 ligand (CD40L, CD154), and LFA-1 (CD18)/ICAM-1 (CD54). T-B cell costimulation also plays a significant role in autoimmune diseases such as systemic lupus erythematosus. Murine HgCl2-induced autoimmunity (mHgIA) is a T cell-dependent systemic autoimmune disease that shares a number of common pathogenic mechanisms with idiopathic lupus. In this report, the significance of costimulation in mHgIA is examined by attempting to induce disease in mice deficient in either CD40L, CD28, or ICAM-1. Unlike absence of ICAM-1, homozygous deficiencies in either CD40L or CD28 significantly reduced the development of mHgIA. CD40L displayed a gene dosage effect as heterozygous mice also showed reduction of autoantibody responses and immunopathology. Markers of T cell activation such as CD44 and CTLA-4 were associated with disease expression in wild-type and ICAM-1-deficient mice but not in CD40L- or CD28-deficient mice. Absence of CTLA-4 expression in CD40L−/− mice suggests that signaling via both CD28 and CD40L is important for T cell activation and subsequent autoimmunity in mHgIA. Attempts to circumvent the absence of CD40L by increasing CD28 signaling via agonistic Ab failed to elicit CTLA-4 expression. These findings indicate that breaking of self-tolerance in mHgIA requires signaling via both the CD28/B7 and CD40/CD40L pathways.
2B4 (CD244) Is Expressed and Functional on Human Eosinophils
Authors: Munitz Ariel , Bachelet Ido , Fraenkel Shira , Katz Gil , Mandelboim Ofer , Simon Hans-Uwe , Moretta Lorenzo , Colonna Marco , Levi-Schaffer Francesca
Published: April 2014

Eosinophils are present in parasitic, allergic, various immunological, and malignant disorders as well as in a variety of idiopathic hypereosinophilic syndromes. However, their exact role in some of these conditions remains elusive. They can be activated both in vivo and in vitro by various agonists, such as Igs, lipid mediators, and cytokines. By phenotyping the surface of the eosinophils, it may be possible to better define their function(s) in different pathophysiological settings. In the present work we screened eosinophils with a panel of Abs recognizing CD2 subfamily receptors usually present on a number of hemopoietic cells. We have demonstrated that human peripheral blood eosinophils, but not basophils or neutrophils, express NTB-A. In addition eosinophils express 2B4, CD84, CD58, and CD48, but not signaling lymphocytic activation molecule or CD2, on their surface (FACS). Cross-linking of 2B4 on eosinophils elicited a significant release of eosinophil peroxidase (30 min), IFN-γ, and IL-4 (18 h). Moreover, activation of eosinophils via 2B4 induced eosinophil-mediated cytotoxicity toward two malignant cell lines, i.e., mouse mastocytoma P815 and EBV-infected 721.221 B cell lines. Cross-linking of 2B4 on the surface of eosinophils or pervenadate treatment elicited ERK and tyrosine phosphorylation, respectively. Furthermore, we showed that eosinophils express slam-associated protein. The demonstration that human eosinophils express a functional 2B4 receptor indicates a broader role for these cells in health and disease.
Dysfunctional Blood and Target Tissue CD4+CD25high Regulatory T Cells in Psoriasis: Mechanism Underlying Unrestrained Pathogenic Effector T Cell Proliferation
Authors: Sugiyama Hideaki , Gyulai Rolland , Toichi Eiko , Garaczi Edina , Shimada Shinji , Stevens Seth R. , McCormick Thomas S. , Cooper Kevin D.
Published: April 2014

The balance between regulatory and effector functions is important for maintaining efficient immune responses, while avoiding autoimmunity. The inflammatory skin disease psoriasis is sustained by the ongoing activation of pathogenic effector T cells. We found that a CD4+ T lymphocyte subpopulation in peripheral blood, phenotypically CD25high, CTLA-4+, Foxp3high (regulatory T (Treg) cells), is deficient in its suppressor activity in psoriasis. This was associated with accelerated proliferation of CD4+ responder T cells in psoriasis, the majority of which expressed CXCR3. Nevertheless, criss-cross experiments isolated the defect to psoriatic Treg cells. To examine Treg cells in a nonlymphoid tissue of a human T cell-mediated disease, Treg cells were also analyzed and isolated from the site of inflammation, psoriatic lesional skin. At the regulatory vs effector T cells ratios calculated to be present in skin, however, the psoriatic Treg cell population demonstrated decreased suppression of effector T cells. Thus, dysfunctional blood and target tissue CD4+CD25high Treg cell activity may lead to reduced restraint and consequent hyperproliferation of psoriatic pathogenic T cells in vivo. These findings represent a critical component of human organ-specific autoimmune disease and may have important implications with regard to the possible therapeutic manipulation of Treg cells in vivo.
Cellular Interactions in Lymph Node Development
Authors: Cupedo Tom , Mebius Reina E.
Published: April 2014

The organized accumulation of lymphocytes is a biological phenomenon used to optimize both homeostatic immune surveillance, as well as chronic responses to pathogenic stimuli. During embryonic development, circulating hemopoietic cells gather at predestined sites throughout the body, where they are subsequently arranged in T and B cell-specific areas characteristic of secondary lymphoid organs. In contrast, the body seems to harbor a limited second set of selected sites that support formation of organized lymphoid aggregates. However, these are only revealed at times of local, chronic inflammation, when so-called tertiary lymphoid structures appear. Once thought of as two distinct phenomena, recent insights suggest that highly similar networks of paracrine interactions regulate the formation of both secondary and tertiary lymphoid structures. This review will focus on these cellular interactions between organizing and inducing cell populations leading to the formation of lymph nodes or organized inflammatory infiltrates.
Differential Roles of Vascular Endothelial Growth Factor Receptors 1 and 2 in Dendritic Cell Differentiation
Authors: Dikov Mikhail M. , Ohm Joyce E. , Ray Neelanjan , Tchekneva Elena E. , Burlison Jared , Moghanaki Drew , Nadaf Sorena , Carbone David P.
Published: April 2014

Impaired Ag-presenting function in dendritic cells (DCs) due to abnormal differentiation is an important mechanism of tumor escape from immune control. A major role for vascular endothelial growth factor (VEGF) and its receptors, VEGFR1/Flt-1 and VEGFR2/KDR/Flk-1, has been documented in hemopoietic development. To study the roles of each of these receptors in DC differentiation, we used an in vitro system of myeloid DC differentiation from murine embryonic stem cells. Exposure of wild-type, VEGFR1−/−, or VEGFR2−/− embryonic stem cells to exogenous VEGF or the VEGFR1-specific ligand, placental growth factor, revealed distinct roles of VEGF receptors. VEGFR1 is the primary mediator of the VEGF inhibition of DC maturation, whereas VEGFR2 tyrosine kinase signaling is essential for early hemopoietic differentiation, but only marginally affects final DC maturation. SU5416, a VEGF receptor tyrosine kinase inhibitor, only partially rescued the mature DC phenotype in the presence of VEGF, suggesting the involvement of both tyrosine kinase-dependent and independent inhibitory mechanisms. VEGFR1 signaling was sufficient for blocking NF-κB activation in bone marrow hemopoietic progenitor cells. VEGF and placental growth factor affect the early stages of myeloid/DC differentiation. The data suggest that therapeutic strategies attempting to reverse the immunosuppressive effects of VEGF in cancer patients might be more effective if they specifically targeted VEGFR1.