bluuebunny/crossref_metadata_2025 huggingface dataset:
A Schistosome-Expressed Immunomodulatory Glycoconjugate Expands Peritoneal Gr1+ Macrophages That Suppress Naive CD4+ T Cell Proliferation Via an IFN-γ and Nitric Oxide-Dependent Mechanism
Authors: Atochina Olga , Daly-Engel Toby , Piskorska Danuta , McGuire Edward , Harn Donald A.
Published: April 2014

Lacto-N-fucopentaose III (LNFPIII) is found in human milk and on the Th2 driving helminth parasite Schistosoma mansoni. This pentasaccharide drives Th2-type responses in vivo and in vitro when conjugated to a carrier. In an attempt to further understand early events in Th1 to Th2 switching, we examined phenotypic and functional changes in peritoneal cell populations in BALB/c and SCID mice following LNFPIII-dextran injection. We found that i.p. injection with LNFPIII-dextran resulted in rapid (<20 h) expansion of the Gr1+ subpopulation of F4/80+/CD11b+ peritoneal cells, comprising up to 75% of F4/80+/CD11b+ peritoneal cells compared with 18% in uninjected or dextran-injected mice. Functionally, these cells suppressed anti-CD3- and anti-CD28-induced proliferation of naive CD4+ T cells. LNFPIII-dextran also expanded functional Gr1+ suppressor macrophages in SCID mice, demonstrating that expansion and function of suppressor cells did not require T cells. Suppression in both BALB/c and SCID mice was NO and IFN-γ dependent, as addition of inhibitors of inducible NO synthase (NG-monomethyl-l-arginine), as well as anti-IFN-γ Abs, restored the ability of CD4+ T cells to proliferate in vitro. Depletion of the F4/80+ subset of Gr1+ cells eliminated the suppressive activity of peritoneal exudate cells showing that these cells were macrophages. Thus, LNFPIII-dextran rapidly expands the Gr1+ suppressor macrophage population in the peritoneal cavities of otherwise naive mice. These Gr1+ cells suppress proliferation of naive CD4+ T cells in an NO-dependent mechanism, and may play a regulatory role in the switching of Th1- to Th2-type responses.
βc Cytokine Receptor-Induced Stimulation of cAMP Response Element Binding Protein Phosphorylation Requires Protein Kinase C In Myeloid Cells: A Novel Cytokine Signal Transduction Cascade
Authors: Gubina Elaina , Luo Xu , Kwon E. , Sakamoto Kathleen , Shi Yu Fang , Mufson R. Allan
Published: April 2014

We have recently shown that IL-3R occupancy activates a phosphatidylcholine-specific phospholipase C, and the sustained diacylglycerol accumulation subsequently activates protein kinase C (PKC). In human IL-3-dependent myeloid cells (TF-1), the novel PKCε isoform regulates bcl-2 expression and cell survival. The report of a PKC activatable cAMP response element (CRE) in the bcl-2 promoter and a role for PKC in bcl-2 expression in B cells led us to the hypothesis that PKC phosphorylation activates transcription factor CREB after IL-3R engagement. We found that IL-3 and GM-CSF induced phosphorylation of CREB on Ser133 in TF-1 cells, and this phosphorylation was blocked by two structurally unrelated classes of PKC inhibitors. An inhibitor of cyclic nucleotide-dependent kinases did not block this phosphorylation. IL-4, which is biologically active in these cells but does not use the β common subunit, did not phosphorylate CREB on Ser133. Inhibition of mitogen-activated protein kinase kinase activity also inhibited IL3-induced CREB phosphorylation. The PKC inhibitors, but not a cyclic nucleotide-dependent kinase inhibitor, blocked IL-3 activation of CRE-dependent transcription from an egr-1 promoter/chloramphenicol acetyltransferase (CAT) reporter construction transiently transfected into TF-1 cells. Finally, TF-1 cells stably overexpressing PKCε, but not the δ isoform of PKC, enhanced CRE-dependent CAT expression from the promoter/reporter construction. Therefore, it is likely that a PKCε kinase cascade resulting in CREB phosphorylation represents a novel signal transduction cascade for regulating cellular gene expression through the β common cytokine receptor.
CD4 Promotes Breadth in the TCR Repertoire
Authors: Wang Qi , Malherbe Laurent , Zhang DongJi , Zingler Kurt , Glaichenhaus Nicolas , Killeen Nigel
Published: April 2014

A diverse population of MHC class II-restricted CD4 lineage T cells develops in mice that lack expression of the CD4 molecule. In this study, we show that the TCR repertoire selected in the absence of CD4 is distinct, but still overlapping in its properties with that selected in the presence of CD4. Immunization of mice lacking CD4 caused the clonal expansion of T cells that showed less breadth in the range of Ag-binding properties exhibited by their TCRs. Specifically, the CD4-deficient Ag-specific TCR repertoire was depleted of TCRs that demonstrated low-affinity binding to their ligands. The data thus suggest a key role for CD4 in broadening the TCR repertoire by potentiating productive TCR signaling and clonal expansion in response to the engagement of low-affinity antigenic ligands.
Enhanced Immune Responses in Transgenic Mice Expressing a Truncated Form of the Lymphocyte Semaphorin CD100
Authors: Watanabe Chie , Kumanogoh Atsushi , Shi Wei , Suzuki Kazuhiro , Yamada Shuichi , Okabe Masaru , Yoshida Kanji , Kikutani Hitoshi
Published: April 2014

CD100/Sema4D is a 150-kDa transmembrane protein that belongs to the semaphorin family. Binding of CD100 to CD72 enhances the immune response by turning off the negative signaling effects of CD72. To investigate the physiological functions of CD100 in vivo, we generated transgenic mice expressing a truncated form of CD100. A large amount of the soluble form of CD100 was detected in the sera of mice expressing a truncated form of CD100, although the amount of CD100 was only slightly elevated on the surface of B cells. In the mutant mice the development of conventional B and T cells appeared normal in terms of the surface marker phenotypes, while the number of CD5+ B-1 cells in the peritoneal cavity increased in comparison with wild-type mice. In vitro proliferation and Ig production of B cells in response to CD40 stimulation were considerably enhanced in mice expressing a truncated form of CD100. Additionally, in vivo both Ab responses against T cell-dependent Ags and generation of Ag-specific T cells were enhanced. Furthermore, introduction of the CD100-transgene could restore in vitro B cell responses as well as in vivo Ab production against T cell-dependent Ag in CD100-deficient mice. Collectively, these results not only indicate that CD100 has an important role in the immune system, but also that the soluble form of CD100 released from the cell surface can exert functions in vivo.
Overcoming the Signaling Defect of Lyn-Sequestering, Signal-Curtailing FcεRI Dimers: Aggregated Dimers Can Dissociate from Lyn and Form Signaling Complexes with Syk
Authors: Lara Martha , Ortega Enrique , Pecht Israel , Pfeiffer Janet R. , Martinez A. Marina , Lee Rebecca J. , Surviladze Zurab , Wilson Bridget S. , Oliver Janet M.
Published: April 2014

Clustering the tetrameric (αβγ2) IgE receptor, FcεRI, on basophils and mast cells activates the Src-family tyrosine kinase, Lyn, which phosphorylates FcεRI β and γ subunit tyrosines, creating binding sites for the recruitment and activation of Syk. We reported previously that FcεRI dimers formed by a particular anti-FcεRI α mAb (H10) initiate signaling through Lyn activation and FcεRI subunit phosphorylation, but cause only modest activation of Syk and little Ca2+ mobilization and secretion. Curtailed signaling was linked to the formation of unusual, detergent-resistant complexes between Lyn and phosphorylated receptor subunits. Here, we show that H10-FcεRI multimers, induced by adding F(ab′)2 of goat anti-mouse IgG to H10-treated cells, support strong Ca2+ mobilization and secretion. Accompanying the recovery of signaling, H10-FcεRI multimers do not form stable complexes with Lyn and do support the phosphorylation of Syk and phospholipase Cγ2. Immunogold electron microscopy showed that H10-FcεRI dimers colocalize preferentially with Lyn and are rarely within the osmiophilic “signaling domains” that accumulate FcεRI and Syk in Ag-treated cells. In contrast, H10-FcεRI multimers frequently colocalize with Syk within osmiophilic patches. In sucrose gradient centrifugation analyses of detergent-extracted cells, H10-treated cells show a more complete redistribution of FcεRI β from heavy (detergent-soluble) to light (Lyn-enriched, detergent-resistant) fractions than cells activated with FcεRI multimers. We hypothesize that restraints imposed by the particular orientation of H10-FcεRI dimers traps them in signal-initiating Lyn microdomains, and that converting the dimers to multimers permits receptors to dissociate from Lyn and redistribute to separate membrane domains that support Syk-dependent signal propagation.
Epitope Mapping of the Indirect T Cell Response to Allogeneic Class I MHC: Sequences Shared by Donor and Recipient MHC May Prime T Cells That Provide Help for Alloantibody Production
Authors: Lovegrove Emma , Pettigrew Gavin J. , Bolton Eleanor M. , Bradley J. Andrew
Published: April 2014

Indirect allorecognition occurs when T cells recognize donor MHC presented as peptide epitopes by recipient APC, but the precise nature of the epitopes involved remains unclear. Rejection of rat MHC class I-disparate PVG.R8 (RT1.Aa) grafts by PVG.RT1u (RT1.Au) recipients is mediated by indirectly restricted CD4 T cells that provide help for the generation of alloantibody. In this study, epitope mapping was performed using a functionally relevant readout (alloantibody production) to identify key peptides that prime an indirect alloimmune response, leading to graft rejection. PVG.RT1u rats were immunized with a series of overlapping 15-mer peptides (peptides 1–18) that spanned the α1 and α2 domains of the RT1.Aa molecule. Several peptides were able to accelerate both the alloantibody response to the intact RT1.Aa Ag and PVG.R8 heart graft rejection. An immunodominant epitope was identified within the hypervariable region of the α1 domain. Fine mapping of this region with a second series of peptides overlapping by single amino acids confirmed the presence of an eight-amino acid core determinant. Additional “subdominant” epitopes were identified, two of which were located within regions of amino acid homology between the RT1.Aa and RT1.Au molecules and not, as had been expected, within other hypervariable regions. The contribution of self-epitopes to indirect allorecognition was emphasized by the demonstration that i.v. administration of a 15-mer peptide encompassing one of the subdominant self-determinants diminished the recipient’s ability to mount an alloantibody response on challenge with intact Aa alloantigen. Our findings suggest that cryptic self-epitopes recognized by autoreactive T cells may contribute to allograft rejection and should be considered when designing novel strategies for inducing tolerance to alloantigen.
Cytokines Regulate the Capacity of CD8α+ and CD8α− Dendritic Cells to Prime Th1/Th2 Cells In Vivo
Authors: Maldonado-López Roberto , Maliszewski Charlie , Urbain Jacques , Moser Muriel
Published: April 2014

Prior studies have shown that subclasses of dendritic cells (DC) direct the development of distinct Th populations in rodents and in humans. In the mouse, we have recently shown that administration of Ag-pulsed CD8α− DC induces a Th2-type response, whereas injection of CD8α+ DC leads to Th1 differentiation. To define the DC-derived factors involved in the polarization of Th responses, we injected either subset purified from mice genetically deficient for IFN-γ, IL-4, IL-12, or IL-10 into wild-type animals. In this work, we report that DC-derived IL-12 and IFN-γ are required for Th1 priming by CD8α+ DC, whereas IL-10 is required for optimal development of Th2 cells by CD8α− DC. The level of IL-12 produced by the DC appears to determine the Th1/Th2 balance in vivo. We further show that the function of DC subsets displays some flexibility. Treatment of DC with IL-10 in vitro induces a selective decrease in the viability of CD8α+ DC. Conversely, incubation with IFN-γ down-regulates the Th2-promoting capacities of CD8α− DC and increases the Th1-skewing properties of both subsets.
Impaired Activation of Islet-Reactive CD4 T Cells in Pancreatic Lymph Nodes of B Cell-Deficient Nonobese Diabetic Mice
Authors: Greeley Siri Atma W. , Moore Daniel J. , Noorchashm Hooman , Noto Lauren E. , Rostami Susan Y. , Schlachterman Alexander , Song Howard K. , Koeberlein Brigitte , Barker Clyde F. , Naji Ali
Published: April 2014

Despite the impressive protection of B cell-deficient (μMT−/−) nonobese diabetic (NOD) mice from spontaneous diabetes, existence of mild pancreatic islet inflammation in these mice indicates that initial autoimmune targeting of β cells has occurred. Furthermore, μMT−/− NOD mice are shown to harbor a latent repertoire of diabetogenic T cells, as evidenced by their susceptibility to cyclophosphamide-induced diabetes. The quiescence of this pool of islet-reactive T cells may be a consequence of impaired activation of T lymphocytes in B cell-deficient NOD mice. In this regard, in vitro anti-CD3-mediated stimulation demonstrates impaired activation of lymph node CD4 T cells in μMT−/− NOD mice as compared with that of wild-type counterparts, a deficiency that is correlated with an exaggerated CD4 T cell:APC ratio in lymph nodes of μMT−/− NOD mice. This feature points to an insufficient availability of APC costimulation on a per T cell basis, resulting in impaired CD4 T cell activation in lymph nodes of μMT−/− NOD mice. In accordance with these findings, an islet-reactive CD4 T cell clonotype undergoes suboptimal activation in pancreatic lymph nodes of μMT−/− NOD recipients. Overall, the present study indicates that B cells in the pancreatic lymph node microenvironment are critical in overcoming a checkpoint involving the provision of optimal costimulation to islet-reactive NOD CD4 T cells.
Spatial Organization of Signal Transduction Molecules in the NK Cell Immune Synapses During MHC Class I-Regulated Noncytolytic and Cytolytic Interactions
Authors: Vyas Yatin M. , Mehta Kamini M. , Morgan Margaret , Maniar Hina , Butros Linda , Jung Steffen , Burkhardt Janis K. , Dupont Bo
Published: April 2014

The cytolytic activity of NK cells is tightly regulated by inhibitory receptors specific for MHC class I Ags. We have investigated the composition of signal transduction molecules in the supramolecular activation clusters in the MHC class I-regulated cytolytic and noncytolytic NK cell immune synapses. KIR2DL3-positive NK clones that are specifically inhibited in their cytotoxicity by HLA-Cw*0304 and polyclonal human NK cells were used for conjugate formation with target cells that are either protected or are susceptible to NK cell-mediated cytotoxicity. Polarization of talin, microtubule-organizing center, and lysosomes occurred only during cytolytic interactions. The NK immune synapses were analyzed by three-dimensional immunofluorescence microscopy, which showed two distinctly different synaptic organizations in NK cells during cytolytic and noncytolytic interactions. The center of a cytolytic synapse with MHC class I-deficient target is comprised of a complex of signaling molecules including Src homology (SH)2-containing protein tyrosine phosphatase-1 (SHP-1). Closely related molecules with overlapping functions, such as the Syk kinases, SYK, and ZAP-70, and adaptor molecules, SH2 domain-containing leukocyte protein of 76 kDa and B cell linker protein, are expressed in activated NK cells and are all recruited to the center of the cytolytic synapse. In contrast, the noncytolytic synapse contains SHP-1, but is lacking other components of the central supramolecular activation cluster. These findings indicate a functional role for SHP-1 in both the cytolytic and noncytolytic interactions. We also demonstrate, in three-cell conjugates, that a single NK cell forms a cytolytic synapse with a susceptible target cell in the presence of both susceptible and nonsusceptible target cells.
Protection from Pulmonary Fibrosis in the Absence of CCR2 Signaling
Authors: Moore Bethany B. , Paine Robert , Christensen Paul J. , Moore Thomas A. , Sitterding Stephanie , Ngan Rose , Wilke Carol A. , Kuziel William A. , Toews Galen B.
Published: April 2014

Pulmonary fibrosis can be modeled in animals by intratracheal instillation of FITC, which results in acute lung injury, inflammation, and extracellular matrix deposition. We have previously shown that despite chronic inflammation, this model of pulmonary fibrosis is lymphocyte independent. The CC chemokine monocyte-chemoattractant protein-1 is induced following FITC deposition. Therefore, we have investigated the contribution of the main monocyte-chemoattractant protein-1 chemokine receptor, CCR2, to the fibrotic disease process. We demonstrate that CCR2−/− mice are protected from fibrosis in both the FITC and bleomycin pulmonary fibrosis models. The protection is specific for the absence of CCR2, as CCR5−/− mice are not protected. The protection is not explained by differences in acute lung injury, or the magnitude or composition of inflammatory cells. FITC-treated CCR2−/− mice display differential patterns of cellular activation as evidenced by the altered production of cytokines and growth factors following FITC inoculation compared with wild-type controls. CCR2−/− mice have increased levels of GM-CSF and reduced levels of TNF-α compared with FITC-treated CCR2+/+ mice. Thus, CCR2 signaling promotes a profibrotic cytokine cascade following FITC administration.
Endothelial Cell Costimulation of T Cell Activation Through CD58-CD2 Interactions Involves Lipid Raft Aggregation
Authors: Mestas Javier , Hughes Christopher C. W.
Published: April 2014

Human endothelial cells (EC) costimulate CD4+ memory T cell activation through CD58-CD2 interactions. In this study we tested the hypothesis that EC activate distinct costimulatory pathways in T cells that target specific transcription factors. AP-1, composed of fos and jun proteins, is a critical effector of TCR signaling and binds several sites in the IL-2 promoter. EC augment c-fos promoter activity in T cells; however, deletion analysis reveals no transcription factor binding sites in the promoter uniquely responsive to EC costimulation. Overexpression of AP-1 proteins in T cells augments the activity of an AP-1-luciferase reporter gene equally in the absence or the presence of EC costimulation. Interestingly, EC stimulate a similar 2- to 3-fold up-regulation of AP-1, NF-AT, NF-κB, and NF-IL-2-luciferase reporters. CD2 mAbs completely block EC effects on all of these pathways, as well as costimulation of IL-2 secretion. We conclude that EC costimulation through CD2 does not trigger a single distinct costimulatory pathway in T cells, but rather, it amplifies several pathways downstream of the TCR. Indeed, we find that early EC costimulation acts “upstream” of the TCR by promoting lipid raft aggregation, thus amplifying TCR signaling. Soluble CD2 mAbs block EC-induced raft aggregation, whereas cross-linking CD2 promotes aggregation. These data are consistent with the critical role of CD2 in organizing the T cell-APC contact zone.
Rudimentary TCR Signaling Triggers Default IL-10 Secretion by Human Th1 Cells
Authors: Burrows Gregory G. , Chou Yuan K. , Wang Chunhe , Chang Justin W. , Finn Thomas P. , Culbertson Nicole E. , Kim Joseph , Bourdette Dennis N. , Lewinsohn Deborah A. , Lewinsohn David M. , Ikeda Masayuki , Yoshioka Tohru , Allen Charles N. , Offner Halina , Vandenbark Arthur A.
Published: April 2014

Understanding the process of inducing T cell activation has been hampered by the complex interactions between APC and inflammatory Th1 cells. To dissociate Ag-specific signaling through the TCR from costimulatory signaling, rTCR ligands (RTL) containing the α1 and β1 domains of HLA-DR2b (DRA*0101:DRB1*1501) covalently linked with either the myelin basic protein peptide 85–99 (RTL303) or CABL-b3a2 (RTL311) peptides were constructed to provide a minimal ligand for peptide-specific TCRs. When incubated with peptide-specific Th1 cell clones in the absence of APC or costimulatory molecules, only the cognate RTL induced partial activation through the TCR. This partial activation included rapid TCR ζ-chain phosphorylation, calcium mobilization, and reduced extracellular signal-related kinase activity, as well as IL-10 production, but not proliferation or other obvious phenotypic changes. On restimulation with APC/peptide, the RTL-pretreated Th1 clones had reduced proliferation and secreted less IFN-γ; IL-10 production persisted. These findings reveal for the first time the rudimentary signaling pattern delivered by initial engagement of the external TCR interface, which is further supplemented by coactivation molecules. Activation with RTLs provides a novel strategy for generating autoantigen-specific bystander suppression useful for treatment of complex autoimmune diseases.
Immunobiological Analysis of TCR Single-Chain Transgenic Mice Reveals New Possibilities for Interaction between CDR3α and an Antigenic Peptide Bound to MHC Class I
Authors: Zhang Weijia , Honda Shinichiro , Wang Fuming , DiLorenzo Teresa P. , Kalergis Alexis M. , Ostrov David A. , Nathenson Stanley G.
Published: April 2014

The interaction between TCRs and peptides presented by MHC molecules determines the specificity of the T cell-mediated immune response. To elucidate the biologically important structural features of this interaction, we generated TCR β-chain transgenic mice using a TCR derived from a T cell clone specific for the immunodominant peptide of vesicular stomatitis virus (RGYVYQGL, VSV8) presented by H-2Kb. We immunized these mice with VSV8 or analogs substituted at TCR contact residues (positions 1, 4, and 6) and analyzed the CDR3α sequences of the elicited T cells. In VSV8-specific CTLs, we observed a highly conserved residue at position 93 of CDR3α and preferred Jα usage, indicating that multiple residues of CDR3α are critical for recognition of the peptide. Certain substitutions at peptide position 4 induced changes at position 93 and in Jα usage, suggesting a potential interaction between CDR3α and position 4. Cross-reactivity data revealed the foremost importance of the Jα region in determining Ag specificity. Surprisingly, substitution at position 6 of VSV8 to a negatively charged residue induced a change at position 93 of CDR3α to a positively charged residue, suggesting that CDR3α may interact with position 6 in certain circumstances. Analogous interactions between the TCR α-chain and residues in the C-terminal half of the peptide have not yet been revealed by the limited number of TCR/peptide-MHC crystal structures reported to date. The transgenic mouse approach allows hundreds of TCR/peptide-MHC interactions to be examined comparatively easily, thus permitting a wide-ranging analysis of the possibilities for Ag recognition in vivo.
CXCR3 Expression on CD34+ Hemopoietic Progenitors Induced by Granulocyte-Macrophage Colony-Stimulating Factor: II. Signaling Pathways Involved
Authors: Jinquan Tan , Anting Liu , Jacobi Henrik H. , Glue Christian , Jing Chen , Ryder Lars P. , Madsen Hans O. , Svejgaard Arne , Skov Per S. , Malling Hans-Jørgen , Poulsen Lars K.
Published: April 2014

CXCR3, known to have four ligands (IFN-γ inducible protein 10 (γ IP-10), monokine induced by IFN-γ (Mig), I-TAC, and 6Ckine), is predominately expressed on memory/activated T lymphocytes. We recently reported that GM-CSF induces CXCR3 expression on CD34+ hemopoietic progenitors, in which γ IP-10 and Mig induce chemotaxis and adhesion. Here we further report that stimulation with GM-CSF causes phosphorylation of Syk protein kinase, but neither Casitas B-lineage lymphoma (Cbl) nor Cbl-b in CD34+ hemopoietic progenitors can be blocked by anti-CD116 mAb. Specific Syk blocking generated by PNA antisense completely inhibits GM-CSF-induced CXCR3 expression in CD34+ progenitors at both mRNA and protein as well as at functional levels (chemotaxis and adhesion). Cbl and Cbl-b blocking have no such effects. Thus, GM-CSF binds to its receptor CD116, and consequently activates Syk phosphorylation, which leads to induce CXCR3 expression. γ IP-10 and Mig can induce Syk, Cbl, and Cbl-b phosphorylation in CD34+ progenitors by means of CXCR3. γ IP-10 or Mig has induced neither chemotaxis nor adhesion in GM-CSF-stimulated Cbl-b-blocked CD34+ hemopoietic progenitors, whereas SDF-1α induces both chemotaxis and adhesion in these cells. Interestingly, γ IP-10 and Mig can induce chemotaxis and adhesion in GM-CSF-stimulated Syk- or Cbl-blocked CD34+ hemopoietic progenitors. Thus, Cbl-b, but not Syk and Cbl phosphorylation, is essential for γ IP-10- and Mig-induced chemotaxis and adhesion in CD34+ hemopoietic progenitors. This study provides a useful insight into novel signaling transduction pathways of the functions of CXCR3/γ IP-10 and Mig, which may be especially important in the cytokine/chemokine environment for mobilization, homing, and recruitment during proliferation, differentiation, and maturation of hemopoietic progenitor cells.
The Cell Type-Specific Expression of the Murine IL-13 Gene Is Regulated by GATA-3
Authors: Kishikawa Hiroko , Sun Jenny , Choi Andrew , Miaw Shi-Chuen , Ho I-Cheng
Published: April 2014

IL-13, a Th2 cell-specific cytokine, is a major effector molecule mediating several pathological features of allergic asthma. However, the transcriptional regulation of the IL-13 gene remains unclear. Here we demonstrate, by using intracellular cytokine staining, that IL-13 is not always coexpressed with other Th2 cytokines in normal Th cells on a single cell basis. In addition, we identified and cloned a minimal inducible and cell type-specific promoter of the murine IL-13 gene. The cell type specificity of the minimal IL-13 promoter is mediated by a functionally critical GATA-3 site that binds endogenous GATA-3 proteins, whereas the induction by PMA/ionomycin is mediated by distinct cis-acting elements. Furthermore, by expressing GATA-3 in wild-type and c-maf transgenic Th1 cells, we demonstrate that the expression of IL-13 is regulated by a mechanism distinct from that regulating the expression of IL-4, and that the expression of Th1 and Th2 cytokine genes does not have to be mutually exclusive in effector Th cells.
Unique Function for Carboxyl-Terminal Domain of Oct-2 in Ig-Secreting Cells
Authors: Sharif M. Nusrat , Radomska Hanna S. , Miller David M. , Eckhardt Laurel A.
Published: April 2014

The activity of Ig gene promoters and enhancers is regulated by two related transcription factors, Oct-1 (ubiquitous) and Oct-2 (B lineage specific), which bind the octamer motif (ATTTGCAT) present in these elements. As Ig promoter-binding factors, Oct-1 and Oct-2 each work together with a B lymphocyte-specific cofactor OCA-B/OBF-1/Bob-1 that interacts with them through their POU (DNA-binding) domains. Because both can mediate Ig promoter activity in B cells, there has been some question as to whether these two octamer-binding factors serve distinct functions in lymphocytes. We have shown previously that the silencing of B lymphocyte-specific genes in plasmacytoma × T lymphoma hybrids can be prevented by preserving Oct-2 expression. The pronounced effect of this transcription factor on the phenotype of plasmacytoma × T lymphoma hybrids established a critical role for Oct-2 not only in maintaining Ig gene expression, but in maintaining the overall genetic program of Ig-secreting cells. In the present study, we have explored the functional differences between Oct-1 and Oct-2 using chimeric Oct-1/Oct-2 proteins in cell fusion assays. Our results provide further evidence for an essential role for Oct-2 in Ig-secreting cells and identify the C-terminal domain of Oct-2 as responsible for its unique function in these cells.
Identification of a Novel Cytokine, ML-1, and Its Expression in Subjects with Asthma
Authors: Kawaguchi Mio , Onuchic Luiz F. , Li Xiao-Dong , Essayan David M. , Schroeder John , Xiao Hui-Qing , Liu Mark C. , Krishnaswamy Guha , Germino Gregory , Huang Shau-Ku
Published: April 2014

A novel gene, designated ML-1, was identified from a human genomic DNA clone and human T cell cDNA sequences. The second exon of ML-1 gene shares significant sequence identity with the gene encoding IL-17 (IL-17). ML-1 gene expression was up-regulated in activated PBMCs, CD4+ T cells, allergen-specific Th0, Th1, and Th2 clones, activated basophils, and mast cells. Increased expression of the ML-1 gene, but not IL-17, was seen following allergen challenge in four asthmatic subjects, suggesting its role in allergic inflammatory responses. ML-1 from transiently transfected COS-7 cells was able to induce gene expression and protein production for IL-6 and IL-8 (at 10 ng/ml of ML-1: for IL-6, 599.6 ± 19.1 pg/ml; for IL-8, 1724.2 ± 132.9 pg/ml; and at 100 ng/ml of ML-1: for IL-6, 1005.3 ± 55.6 pg/ml; for IL-8, 4371.4 ± 280.5 pg/ml; p < 0.05 for both doses vs baseline) in primary bronchial epithelial (PBE) cells. Furthermore, increased expression of ICAM-1 was found in ML-1-stimulated PBE cells (mean fluorescence intensity (MFI) = 31.42 ± 4.39 vs baseline, MFI = 12.26 ± 1.77, p < 0.05), a functional feature distinct from IL-17 (MFI = 11.07 ± 1.22). This effect was not inhibited by a saturating amount of IL-17. These findings demonstrate that ML-1 is a novel cytokine with a distinct function, and suggest a different receptor for ML-1 on PBE cells.
IL-10 Inhibits Apoptosis of Promyeloid Cells by Activating Insulin Receptor Substrate-2 and Phosphatidylinositol 3′-Kinase
Authors: Zhou Jian-Hua , Broussard Suzanne R. , Strle Klemen , Freund Gregory G. , Johnson Rodney W. , Dantzer Robert , Kelley Keith W.
Published: April 2014

IL-10 is well known to be a potent inhibitor of the synthesis of proinflammatory cytokines, but noninflammatory hemopoietic cells also express IL-10Rs. Here we show that IL-10 directly affects progenitor myeloid cells by protecting them from death following the removal of growth factors. Murine factor-dependent cell progenitors cultured in the absence of growth factors were 43 ± 1% apoptotic after 12 h. Addition of IL-10 at a concentration as low as 100 pg/ml significantly reduced the apoptotic population to 32 ± 3%. At 10 ng/ml, IL-10 caused a 4-fold reduction in the apoptotic population (11 ± 1%). The anti-apoptotic activity of IL-10 was significantly inhibited with a neutralizing IL-10R Ab. Factor-dependent cell progenitor promyeloid cells expressed functional IL-10Rs, as assessed by precipitation of a 110-kDa protein with an Ab to the IL-10R and by the ability of IL-10 to activate Jak1 and Tyk2 and to phosphorylate tyrosine 705 on Stat-3. IL-10 increased tyrosyl phosphorylation of insulin receptor substrate-2 and stimulated the enzymatic activity of both phosphatidylinositol 3′-kinase and Akt. The anti-apoptotic activity of IL-10 was blocked by inhibition of phosphatidylinositol 3′-kinase. Wortmannin and LY294002 also totally inhibited activation of extracellular signal-related kinase (ERK)1/2 by IL-10. Direct inhibition of ERK1/2 with the mitogen-activated protein kinase/ERK kinase inhibitor PD98059 partially, but significantly, impaired the anti-apoptotic activity of IL-10. These data establish that activation of the IL-10R promotes survival of progenitor myeloid cells. This survival-promoting activity is totally due to IL-10 stimulating the insulin receptor substrate-2/PI 3-kinase/Akt pathway, which increases the anti-apoptotic activity of ERK1/2.
A Region of Tapasin That Affects Ld Binding and Assembly
Authors: Turnquist Hēth R. , Vargas Shanna E. , Reber Adrian J. , McIlhaney Mary M. , Li Suling , Wang Ping , Sanderson Sam D. , Gubler Brigitte , van Endert Peter , Solheim Joyce C.
Published: April 2014

Tapasin has been shown to stabilize TAP and to link TAP to the MHC class I H chain. Evidence also has been presented that tapasin influences the loading of peptides onto MHC class I. To explore the relationship between the ability of tapasin to bind to TAP and the MHC class I H chain and the ability of tapasin to facilitate class I assembly, we have created novel tapasin mutants and expressed them in 721.220-Ld cells. One mutant has a deletion of nine amino acid residues (tapasin Δ334–342), and the other has amino acid substitutions at positions 334 and 335. In this report we describe the ability of these mutants to interact with Ld and their effects on Ld surface expression. We found that tapasin Δ334–342 was unable to bind to the Ld H chain, and yet it facilitated Ld assembly and expression. Tapasin Δ334–342 was able to bind and stabilize TAP, suggesting that TAP stabilization may be important to the assembly of Ld. Tapasin mutant H334F/H335Y, unlike tapasin Δ334–342, bound to Ld. Expression of tapasin H334F/H335Y in 721.220-Ld reduced the proportion of cell surface open forms of Ld and retarded the migration of Ld from the endoplasmic reticulum. In total, our results indicate that the 334–342 region of tapasin influences Ld assembly and transport.
Differential Evolution and Stability of Epitope-Specific CD8+ T Cell Responses in EBV Infection
Authors: Catalina Michelle D. , Sullivan John L. , Bak Katherine R. , Luzuriaga Katherine
Published: April 2014

Murine models of lymphocytic choriomeningitis virus infection suggest that the memory CD8+ T cell repertoire is reflective of the CD8+ T cell repertoire generated during acute infection. Less is known regarding the evolution of CD8+ T cell repertoires during human viral infections. We therefore examined epitope-specific CD8+ T cell responses in a large cohort of individuals with acute through latent Epstein-Barr virus infection. Using 16 of 20 published EBV epitopes restricted by HLA-A2, HLA-A3 or HLA-B7, we showed that lytic cycle-specific CD8+ T cell responses predominated during acute EBV infection. However, whereas HLA-A2+-restricted BMLF-1-specific CD8+ T cell responses were maintained through latency, HLA-A2+- and HLA-B7+-restricted BZLF-1, as well as HLA-A3+-restricted BRLF-1 CD8+ T cell responses, were generated but not readily maintained. Analyses of CD8+ T cell responses to EBV latent cycle Ags showed delayed detection and lower frequencies of latent epitope-specific CD8+ T cell responses during acute EBV infection, with maintenance of these responses 1 yr post-EBV infection. Early BMLF-1 and EBNA-3A epitope-specific CD8+ T cell frequencies did not correlate with their frequencies at 1 yr postinfection. Interestingly, populations of EBV-specific CD8+ T cells were stable during 20 mo in our long term EBV-seropositive populations, suggesting homeostasis between virus and the host immune system. This study demonstrates that CD8+ T cell repertoires generated during persistent viral infections are not simply reflective of the initial pool of CD8+ T cells and provides evidence that the generation of CD8+ T cell responses to a persistent infection is a dynamic process.
Notch-1 Regulates NF-κB Activity in Hemopoietic Progenitor Cells
Authors: Cheng Pingyan , Zlobin Andrew , Volgina Veronica , Gottipati Sridevi , Osborne Barbara , Simel Erica J. , Miele Lucio , Gabrilovich Dmitry I.
Published: April 2014

We investigated the interaction between two elements critical for differentiation of hemopoietic cells, the Notch-1 receptor and the transcription factor NF-κB. These factors were studied in hemopoietic progenitor cells (HPC) using Notch-1 antisense transgenic (Notch-AS-Tg) mice. DNA binding of NF-κB as well as its ability to activate transcription was strongly decreased in HPC from Notch-AS-Tg mice. NF-κB-driven transcriptional activity was completely restored after transduction of the cells with retroviral constructs containing activated Notch-1 gene. HPC from Notch-AS-Tg mice have decreased levels of several members of the NF-κB family, p65, p50, RelB, and c-Rel and this is due to down-regulation of the gene expression. To investigate functional consequences of decreased NF-κB activity in transgenic mice, we studied LPS-induced proliferation of B cells and GM-CSF-dependent differentiation of dendritic cells from HPC. These two processes are known to be closely dependent on NF-κB. B cells from Notch-AS-Tg mice had almost 3-fold lower response to LPS than B cells isolated from control mice. Differentiation of dendritic cells was significantly affected in Notch-AS-Tg mice. However, it was restored by transduction of activated Notch-1 into HPC. Taken together, these data indicate that in HPC NF-κB activity is regulated by Notch-1 via transcriptional control of NF-κB.
Enforced Expression of GATA-3 Severely Reduces Human Thymic Cellularity
Authors: Taghon Tom , De Smedt Magda , Stolz Frank , Cnockaert Maggy , Plum Jean , Leclercq Georges
Published: April 2014

Following bone marrow transplantation, patients often suffer from immune incompetence by reduced or late T cell development. Moreover, adult bone marrow stem cells have a lower capacity to generate T cells compared with fetal liver- and umbilical cord blood-derived progenitors. Therefore, enhancing thymic-dependent T cell generation might hold great therapeutic potential. GATA-3 is a transcription factor that is essential in T cell development. In this study we examined the therapeutic potential of GATA-3 to enhance T cell generation by overexpressing GATA-3 in T cell progenitors followed by fetal thymic organ culture (FTOC). We observed that early during FTOC, there was an enhanced differentiation toward the double positive stage of T cell development. From day 10 of FTOC, however, overexpression of GATA-3 induced a severe reduction in thymic cellularity, which probably correlates with the absence of a functional TCR-β chain. We further show that the frequency of apoptosis was increased in GATA-3-transduced thymocytes. Despite the absence of a functional TCR-β chain, GATA-3 transduced progenitors were able to differentiate into CD8β+ double positive thymocytes. This study shows that a strictly regulated expression of GATA-3 is essential for normal T cell development and this puts severe restrictions on the potential therapeutic use of continuously overexpressed GATA-3.
IL-12, STAT4-Dependent Up-Regulation of CD4+ T Cell Core 2 β-1,6-n-Acetylglucosaminyltransferase, an Enzyme Essential for Biosynthesis of P-Selectin Ligands
Authors: Lim Yaw-Chyn , Xie Huijuan , Come Carolyn E. , Alexander Stephen I. , Grusby Michael J. , Lichtman Andrew H. , Luscinskas Francis W.
Published: April 2014

TCR activation of naive T cells in the presence of IL-12 drives polarization toward a Th1 phenotype and synthesis of P- and E-selectin ligands. Fucosyltransferase VII (Fuc-T VII) and core 2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) are critical for biosynthesis of selectin ligands. P-selectin glycoprotein ligand-1 is the best characterized ligand for P-selectin and also binds E-selectin. The contributions of TCR and cytokine signaling pathways to up-regulate Fuc-T VII and C2GnT during biosynthesis of E- and P-selectin ligands, such as P-selectin glycoprotein ligand 1, are unknown. IL-12 signals via the STAT4 pathway. Here, naive DO11.10 TCR transgenic and STAT4−/− TCR transgenic CD4+ T cells were stimulated with Ag and IL-12 (Th1 condition), IL-4 (Th2), or neutralizing anti-IL-4 mAb only (Th0). The levels of Fuc-T VII and C2GnT mRNA in these cells were compared with their adhesive interactions with P- and E-selectin in vitro under flow. The data show IL-12/STAT4 signaling is necessary for induction of C2GnT, but not Fuc-TVII mRNA, and that STAT4−/− Th1 cells do not traffic normally to sites of inflammation in vivo, do not interact with P-selectin, and exhibit a partial reduction of E-selectin interactions under shear stress in vitro. Ag-specific TCR activation in CD4+ T cells was sufficient to trigger induction of Fuc-TVII, but not C2GnT, mRNA and expression of E-selectin, but not P-selectin, ligands. Thus, Fuc-T VII and C2GnT are regulated by different signals during Th cell differentiation, and both cytokine and TCR signals are necessary for the expression of E- and P-selectin ligands.
TCRA Gene Rearrangement in Immature Thymocytes in Absence of CD3, Pre-TCR, and TCR Signaling
Authors: Mancini Stéphane J. C. , Candéias Serge M. , Di Santo James P. , Ferrier Pierre , Marche Patrice N. , Jouvin-Marche Evelyne
Published: April 2014

During thymocyte differentiation, TCRA genes are massively rearranged only after productively rearranged TCRB genes are expressed in association with pTα and CD3 complex molecules within a pre-TCR. Signaling from the pre-TCR via the CD3 complex is thought to be required to promote TCRA gene accessibility and recombination. However, αβ+ thymocytes do develop in pTα-deficient mice, showing that TCRα-chain genes are rearranged, either in CD4−CD8− or CD4+CD8+ thymocytes, in the absence of pre-TCR expression. In this study, we analyzed the TCRA gene recombination status of early immature thymocytes in mutant mice with arrested thymocyte development, deficient for either CD3 or pTα and γc expression. ADV genes belonging to different families were found rearranged to multiple AJ segments in both cases. Thus, TCRA gene rearrangement is independent of CD3 and γc signaling. However, CD3 expression was found to play a role in transcription of rearranged TCRα-chain genes in CD4−CD8− thymocytes. Taken together, these results provide new insights into the molecular control of early T cell differentiation.
Chromatin Remodeling, Measured by a Novel Real-Time Polymerase Chain Reaction Assay, Across the Proximal Promoter Region of the IL-2 Gene
Authors: Rao Sudha , Procko Erik , Shannon M. Frances
Published: April 2014

The structure of chromatin and its remodeling following activation are important aspects of the control of inducible gene transcription. The IL-2 gene is induced in a cell specific-manner in T cells following an antigenic stimulus. We show, using a novel real-time PCR assay, that significant chromatin remodeling of the IL-2 proximal promoter region occurred upon stimulation of both the murine EL-4 T cell line and primary CD4+ T cells. Chromatin remodeling appears to be limited to the first 300 bp of the proximal promoter region as measured by micrococcal nuclease and restriction enzyme accessibility. Time course studies indicated that chromatin remodeling was observed at 1.5 h postinduction and was maintained for up to 16 h. The remodeling is reversible upon removal of the stimulus. The region immediately upstream from the transcription start site, however, remains accessible for up to 16 h. Upon restimulation, remodeling occurs much more rapidly, consistent with a more rapid rise in IL-2 mRNA levels. Using a number of pharmacological inhibitors we show that remodeling is dependent on the presence of specific transcription factors, but not on the modification of histones. The development of this novel chromatin accessibility assay based on real-time PCR has allowed rapid, sensitive, and quantitative measurements on the IL-2 gene following cellular activation in both T cell lines and primary cells.
An Ancient Lectin-Dependent Complement System in an Ascidian: Novel Lectin Isolated from the Plasma of the Solitary Ascidian, Halocynthia roretzi
Authors: Sekine Hideharu , Kenjo Akira , Azumi Kaoru , Ohi Gota , Takahashi Minoru , Kasukawa Reiji , Ichikawa Narumi , Nakata Munehiro , Mizuochi Tsuguo , Matsushita Misao , Endo Yuichi , Fujita Teizo
Published: April 2014

Mannose-binding lectin (MBL) is a C-type lectin involved in the first line of host defense against pathogens and it requires MBL-associated serine protease (MASP) for activation of the complement lectin pathway. To elucidate the origin and evolution of MBL, MBL-like lectin was isolated from the plasma of a urochordate, the solitary ascidian Halocynthia roretzi, using affinity chromatography on a yeast mannan-Sepharose. SDS-PAGE of the eluted proteins revealed a major band of ∼36 kDa (p36). p36 cDNA was cloned from an ascidian hepatopancreas cDNA library. Sequence analysis revealed that the carboxy-terminal half of the ascidian lectin contains a carbohydrate recognition domain (CRD) that is homologous to C-type lectin, but it lacks a collagen-like domain that is present in mammalian MBLs. Purified p36 binds specifically to glucose but not to mannose or N-acetylglucosamine, and it was designated glucose-binding lectin (GBL). The two ascidian MASPs associated with GBL activate ascidian C3, which had been reported to act as an opsonin. The removal of GBL-MASPs complex from ascidian plasma using Ab against GBL inhibits C3-dependent phagocytosis. These observations strongly suggest that GBL acts as a recognition molecule and that the primitive complement system, consisting of the lectin-proteases complex and C3, played a major role in innate immunity before the evolution of an adaptive immune system in vertebrates.
IL-2 During In Vitro Priming Promotes Subsequent Engraftment and Successful Adoptive Tumor Immunotherapy by Persistent Memory Phenotypic CD8+ T Cells
Authors: Bathe Oliver F. , Dalyot-Herman Nava , Malek Thomas R.
Published: April 2014

Adoptive T cell tumor immunotherapy potentially consists of two protective components by the transferred effector cells, the immediate immune response and the subsequent development of memory T cells. The extent by which adoptively transferred CD8+ CTL are destined to become memory T cells is ambiguous as most studies focus on the acute effects on tumor shortly following adoptive transfer. In this study we show that a substantial fraction of the input CTL develop into memory cells that reject a s.c. tumor challenge. The use of exogenous IL-2 or a combination of IL-2 and IL-4, but not solely IL-4, during the ex vivo culture for the CTL inoculation was necessary for efficient development of CD8+ memory T cells. Thus, an important component of adoptive immunotherapy using CTL is the production of CD8+ Ag-specific memory cells which is primarily favored by IL-2 receptor signaling during ex vivo generation of the effector CTL.
The Pulmonary Environment Promotes Th2 Cell Responses After Nasal-Pulmonary Immunization with Antigen Alone, but Th1 Responses Are Induced During Instances of Intense Immune Stimulation
Authors: Jones Harlan P. , Hodge Lisa M. , Fujihashi Kohtaro , Kiyono Hiroshi , McGhee Jerry R. , Simecka Jerry W.
Published: April 2014

The purpose of this study was to determine the nature of the CD4+ Th cell responses induced after nasal-pulmonary immunization, especially those coinciding with previously described pulmonary inflammation associated with the use of the mucosal adjuvant, cholera toxin (CT). The major T cell population in the lungs of naive mice was CD4+, and these cells were shown to be predominantly of Th2 type as in vitro polyclonal stimulation resulted in IL-4, but not IFN-γ, production. After nasal immunization with influenza Ag alone, Th2 cytokine mRNA (IL-4 and IL-5) levels were increased, whereas there was no change in Th1 cytokine (IL-2 and IFN-γ) mRNA expression. The use of the mucosal adjuvant, CT, markedly enhanced pulmonary Th2-type responses; however, there was also a Th1 component to the T cell response. Using in vitro Ag stimulation of pulmonary lymphocytes, influenza virus-specific cytokine production correlated with the mRNA cytokine results. Furthermore, there was a large increase in CD4+ Th cell numbers in lungs after nasal immunization using CT, correlating with the pulmonary inflammatory infiltrate previously described. Coincidentally, both macrophage-inflammatory protein-1α (MIP-1α) and MIP-1β mRNA expression increased in the lungs after immunization with Ag plus CT, while only MIP-1β expression increased when mice were given influenza Ag alone. Our study suggests a mechanism to foster Th1 cell recruitment into the lung, which may impact on pulmonary immune responses. Thus, while Th2 cell responses may be prevalent in modulating mucosal immunity in the lungs, Th1 cell responses contribute to pulmonary defenses during instances of intense immune stimulation.