bluuebunny/crossref_metadata_2025 huggingface dataset:
Cutting Edge: Competition for APC by CTLs of Different Specificities Is Not Functionally Important During Induction of Antiviral Responses
Authors: Probst Hans Christian , Dumrese Tilman , van den Broek Maries F.
Published: April 2014

The hypothesis that T cell competition for access to APC influences priming of CTL responses is a controversial issue. A recent study using OVA as a model Ag supports this hypothesis and received considerable attention. However, using a comparable approach, we reached a different conclusion. We analyzed whether TCR transgenic T cells specific for lymphocytic choriomeningitis virus gp33–41/Db could inhibit the priming of endogenous responses against gp33–41 and against two other lymphocytic choriomeningitis virus glycoprotein-derived CTL epitopes. After priming with different stimuli, gp33–41/Db-specific TCR transgenic T cells reduced the endogenous gp33–41/Db response in a dose-dependent way, but all other endogenous responses were unaffected. Even when >106 TCR transgenic cells were combined with weak priming, no reduction of responses other than of those specific for gp33–41/Db was observed. Thus, competition for APC by CTLs of different specificities is not of functional relevance in antiviral immune responses.
Cutting Edge: Immune Cells as Sources and Targets of the IL-10 Family Members?
Authors: Wolk Kerstin , Kunz Stefanie , Asadullah Khusru , Sabat Robert
Published: April 2014

This study investigated the expression of five novel human IL-10-related molecules and their receptors in blood mononuclear cells. IL-19 and IL-20 were found to be preferentially expressed in monocytes. IL-22 and IL-26 (AK155) expression was exclusively detected in T cells, especially upon type 1 polarization, and in NK cells. IL-24 (melanoma differentiation-associated gene 7) expression was restricted to monocytes and T cells. Detection of these molecules in lymphocytes was predominantly linked to cellular activation. Regarding T cells, IL-26 was primarily produced by memory cells, and its expression was independent on costimulation. In contrast to the high expression of receptors for IL-10 homologs in different tissues and cell lines, monocytes and NK, B, and T cells showed clear expression only of IL-10R1, IL-10R2, and IL-20R2. In these cells, IL-20R2 might be part of a still-unknown receptor complex. Therefore, immune cells may represent a major source but a minor target of the novel IL-10 family members.
Inhibitory Effects of Cytomegalovirus Proteins US2 and US11 Point to Contributions from Direct Priming and Cross-Priming in Induction of Vaccinia Virus-Specific CD8+ T Cells
Authors: Basta Sameh , Chen Weisan , Bennink Jack R. , Yewdell Jonathan W.
Published: April 2014

The extent to which naive CD8+ CTLs (TCD8+) are primed by APCs presenting endogenous Ags (direct priming) or Ags acquired from other infected cells (cross-priming) is a critical topic in basic and applied immunology. To examine the contribution of direct priming in the induction of VV-specific TCD8+, we generated recombinant vaccinia viruses that express human CMV proteins (US2 and US11) that induce the destruction of newly synthesized MHC class I molecules. Expression of US2 or US11 was associated with a 24–63% decrease in numbers of primary or secondary VV-specific TCD8+ responding to i.p. infection. Using HPLC-isolated peptides from VV-infected cells, we show that US2 and US11 selectively inhibit TCD8+ responses to a subset of immunogenic VV determinants. Moreover, VV-US2 and lysates from VV-infected histoincompatible cells elicit TCD8+ specific for a similar subset of VV determinants. These findings indicate that US2 and US11 can function in vivo to interfere with the activation of virus-specific TCD8+. Furthermore, they suggest that 1) both cross-priming and direct priming contribute significantly to the generation of VV-specific TCD8+, 2) the sets of immunogenic vaccinia virus determinants generated by cross-priming and direct priming are not completely overlapping, and 3) cross-priming overrides the effects of cis-acting viral interference with the class I Ag presentation pathway.
Recycling CD1d1 Molecules Present Endogenous Antigens Processed in an Endocytic Compartment to NKT Cells
Authors: Roberts Tonya J. , Sriram Venkataraman , Spence Philip M. , Gui Ming , Hayakawa Kyoko , Bacik Igor , Bennink Jack R. , Yewdell Jonathan W. , Brutkiewicz Randy R.
Published: April 2014

Mouse CD1d1 molecules present endogenous glycolipids to NKT cells. Although glycolipid presentation requires CD1d1 transport through the endocytic pathway, the processing requirements for such endogenous Ag presentation by CD1d1 molecules are undefined. We examined CD1d1 Ag presentation to NKT cells by disrupting endocytic trafficking and function in cells expressing normal and mutated CD1d1 expressed by recombinant vaccinia viruses. Consistent with previous studies, we found that preventing CD1d1 localization to endosomes by altering its cytoplasmic targeting sequences abrogated recognition by Vα14Jα281+ NKT cells without affecting recognition by Vα14− NKT cells. Increasing the pH of acidic compartments by incubating cells with chloroquine or bafilomycin A1 blocked CD1d1 recognition by Vα14+ (but not Vα14−) NKT cells without reducing levels of cell surface CD1d1. Similar results were obtained with primaquine, which interferes with the recycling of cell surface glycoproteins. These results suggest that the loading of a subset of glycolipid ligands onto CD1d1 molecules entails the delivery of cell surface CD1d1 molecules and an acidic environment in the endocytic pathway.
Ligand-Induced TCR Down-Regulation Is Not Dependent on Constitutive TCR Cycling
Authors: Dietrich Jes , Menné Charlotte , Lauritsen Jens Peter H. , von Essen Marina , Rasmussen Anette B. , Ødum Niels , Geisler Carsten
Published: April 2014

TCR internalization takes place both in resting T cells as part of constitutive TCR cycling, after PKC activation, and during TCR triggering. It is still a matter of debate whether these pathways represent distinct pathways. Thus, some studies have indicated that ligand-induced TCR internalization is regulated by mechanisms distinct from those involved in constitutive internalization, whereas other studies have suggested that the ligand-induced TCR internalization pathway is identical with the constitutive pathway. To resolve this question, we first identified requirements for constitutive TCR cycling. We found that in contrast to PKC-induced TCR internalization where both CD3γ-S126 and the CD3γ leucine-based internalization motif are required, constitutive TCR cycling required neither PKC nor CD3γ-S126 but only the CD3γ leucine-based motif. Having identified these requirements, we next studied ligand-induced internalization in cells with abolished constitutive TCR cycling. We found that ligand-induced TCR internalization was not dependent on constitutive TCR internalization. Likewise, constitutive internalization and recycling of the TCR were independent of an intact ligand-induced internalization of the TCR. In conclusion, ligand-induced TCR internalization and constitutive cycling of the TCR represents two independent pathways regulated by different mechanisms.
In Vivo Differentiated Cytokine-Producing CD4+ T Cells Express Functional CCR7
Authors: Debes Gudrun F. , Höpken Uta E. , Hamann Alf
Published: April 2014

Chemokines and their receptors fulfill specialized roles in inflammation and under homeostatic conditions. CCR7 and its ligands, CCL19 and CCL21, are involved in lymphocyte recirculation through secondary lymphoid organs and additionally navigate lymphocytes into distinct tissue compartments. The role of CCR7 in the migration of polarized T effector/memory cell subsets in vivo is still poorly understood. We therefore analyzed murine and human CD4+ cytokine-producing cells developed in vivo for their chemotactic reactivity to CCR7 ligands. The responses of cells producing cytokines, such as IFN-γ, IL-4, and IL-10, as well as of subsets defined by memory or activation markers were comparable to that of naive CD4+ cells, with slightly lower reactivity in cells expressing IL-10 or CD69. This indicates that CCR7 ligands are able to attract naive as well as the vast majority of activated and effector/memory T cell stages. Chemotactic reactivity of these cells toward CCL21 was absent in CCR7-deficient cells, proving that effector cells do not use alternative receptors for this chemokine. Th1 cells generated from CCR7−/− mice failed to enter lymph nodes and Peyer’s patches, but did enter a site of inflammation. These findings indicate that CD4+ cells producing effector cytokines upon stimulation retain the capacity to recirculate through lymphoid tissues via CCR7.
Identification of Naive or Antigen-Experienced Human CD8+ T Cells by Expression of Costimulation and Chemokine Receptors: Analysis of the Human Cytomegalovirus-Specific CD8+ T Cell Response
Authors: Wills Mark R. , Okecha Georgina , Weekes Michael P. , Gandhi Maher K. , Sissons Patrick J. G. , Carmichael Andrew J.
Published: April 2014

Human CMV (HCMV) infection provides an informative model of how long term human CD8+ T cell memory is maintained in the presence of Ag. To clarify the phenotypic identity of Ag-experienced human CD8+ T cells in vivo, we determined the expression of costimulation and chemokine receptors on Ag-specific CD8+ T cells by quantifying individual virus-specific clones in different cell populations using TCR clonotypic probing. In healthy HCMV carriers, expanded CD8+ clones specific for either HCMV tegument protein pp65 or immediate-early protein IE72 are found in both CD45ROhigh cells and the subpopulation of CD45RAhigh cells that lack the costimulatory molecule CD28. In contrast to previous suggested models of CD8+ T cell memory, we found that in healthy virus carriers highly purified CD28−CD45RAhighCCR7− cells are not terminally differentiated, because following stimulation in vitro with specific HCMV peptide these cells underwent sustained clonal proliferation, up-regulated CD45RO and CCR5, and showed strong peptide-specific cytotoxic activity. In an individual with acute primary HCMV infection, HCMV pp65-specific CD8+ T cells are predominantly CD28−CD45ROhighCCR7−. During convalescence, an increasing proportion of pp65-specific CD8+ T cells were CD28−CD45RAhighCCR7−. We conclude that naive human CD8+ T cells are CD28+CD45RAhigh, express CCR7 but not CCR6, and are predominantly CD27+ and L-selectin CD62 ligand-positive. The phenotype CD27+CD45RAhigh should not be used to identify naive human CD8+ T cells, because CD27+CD45RAhigh cells also contain a significant subpopulation of CD28−CD27+ Ag-experienced expanded clones. Thus CD8+ T cell memory to HCMV is maintained by cells of expanded HCMV-specific clones that show heterogeneity of activation state and costimulation molecular expression within both CD45ROhigh and CD28−CD45RAhigh T cell pools.
Reduced Expression of STAT4 and IFN-γ in Macrophages from BALB/c Mice
Authors: Kuroda Etsushi , Kito Tomohiro , Yamashita Uki
Published: April 2014

BALB/c mice have been shown to easily induce Th2 type responses in several infection models. In this study, to examine the mechanisms of Th2 dominant responses in BALB/c mice, we assessed several macrophage functions using C3H/HeN, C57BL/6, and BALB/c mouse strains. Peritoneal macrophages from three strains of mice equally produced IL-12 by stimulation with LPS plus IFN-γ. However, IFN-γ production in response to IL-12 or IL-12 plus IL-18 was much lower in macrophages from BALB/c mice than other strains. IFN-γ produced by activated macrophages induced IL-12R mRNA expression in T cells and macrophages themselves depending on their amount of IFN-γ; namely, macrophages from BALB/c mice induced lower expression of IL-12R. Intracellular levels of STAT4 were much lower in macrophages from BALB/c mice. However, other STATs, such as STAT1 or STAT6, were expressed similarly in the three mouse strains. STAT4 and IFN-γ production by other cell types such as T cells and B cells were equal in C3H/HeN and BALB/c mice. These results indicate that macrophages from Th2-dominant BALB/c mice have different functional characters compared with other mouse strains; that is, STAT4 expression and IFN-γ production are reduced, which is one of the causes to shift to Th2-type responses.
Immune Responses in 4-1BB (CD137)-Deficient Mice
Authors: Kwon Byoung S. , Hurtado Jose C. , Lee Zang H. , Kwack Kyu B. , Seo Su K. , Choi Beom K. , Koller Beverly H. , Wolisi Godwin , Broxmeyer Hal E. , Vinay Dass S.
Published: April 2014

The 4-1BB (a TNFR superfamily member) is an inducible costimulatory molecule that can exert regulatory effects on T cells independently of CD28 stimulation. The in vitro expression of 4-1BB (CD137) is induced following activation of T cells with various stimuli, including anti-TCR mAbs, lectins, and a combination of PMA and ionomycin. To delineate further the physiological role of 4-1BB in immunity, mice deficient in this receptor were generated. These mutant mice developed normally, and were viable and fertile. Humoral responses to vesicular stomatitis virus were comparable with those seen in wild-type mice, whereas the IgG2a and IgG3 isotype responses to keyhole limpet hemocyanin were somewhat reduced in the mutant mice. The 4-1BB-deficient mice demonstrated enhanced T cell proliferation in response to mitogens or anti-CD3 even in the environment of reduced ability to secrete growth-supporting cytokines (IL-2 and IL-4). Although T cells from 4-1BB-deficient mice showed enhanced proliferation, the T cell immune responses of these animals, such as cytokine production and CTL activity, were diminished. In addition, 4-1BB deletion appears to play a role in the regulation of myeloid progenitor cell growth, leading to an increase in these precursor cells in peripheral blood, bone marrow, and spleen.
Vα24-JαQ-Independent, CD1d-Restricted Recognition of α-Galactosylceramide by Human CD4+ and CD8αβ+ T Lymphocytes
Authors: Gadola Stephan D. , Dulphy Nicolas , Salio Mariolina , Cerundolo Vincenzo
Published: April 2014

Human CD1d molecules present an unknown ligand, mimicked by the synthetic glycosphingolipid α-galactosylceramide (αGC), to a highly conserved NKT cell subset expressing an invariant TCR Vα24-JαQ paired with Vβ11 chain (Vα24+Vβ11+ invariant NK T cell (NKTinv)). The developmental pathway of Vα24+Vβ11+NKTinv is still unclear, but recent studies in mice were consistent with a TCR instructive, rather than a stochastic, model of differentiation. Using CD1d-αGC-tetramers, we demonstrate that in humans, TCR variable domains other than Vα24 and Vβ11 can mediate specific recognition of CD1d-αGC. In contrast to Vα24+Vβ11+NKTinv cells, Vα24−/CD1d-αGC-specific T cells express either CD8αβ or CD4 molecules, but they are never CD4 CD8 double negative. We show that CD8αβ+Vα24−/CD1d-αGC-specific T cells exhibit CD8-dependent specific cytotoxicity and have lower affinity TCRs than Vα24+/CD1d-αGC-specific T cells. In conclusion, our results demonstrate that, contrary to the currently held view, recognition of CD1d-αGC complex in humans is not uniformly restricted to the Vα24-JαQ/Vβ11 NKT cell subset, but can be mediated by a diverse range of Vα and Vβ domains. The existence of a diverse repertoire of CD1d-αGC-specific T cells in humans strongly supports their Ag-driven selection.
Differentiation of Human CD8+ T Cells from a Memory to Memory/Effector Phenotype
Authors: Tomiyama Hiroko , Matsuda Tomoko , Takiguchi Masafumi
Published: April 2014

Previous studies of perforin expression and cytokine production in subsets of peripheral human CD45RA−CD8+ T cells with different CD28/CD27 phenotypes showed that CD28+CD45RA−CD8+ and CD27+CD45RA−CD8+ T cells have characteristics of memory T cells, whereas CD28−CD45RA−CD8+ and CD27−CD45RA−CD8+ T cells have characteristics of both memory and effector T cells. However, the differentiation pathway from memory CD8+ T cells into memory/effector CD8+ T cells has not been completely clarified. We investigated this differentiation pathway using EBV- and human CMV (HCMV)-specific CD8+ T cells. Three subsets of CD45RA−CD8+ T cells were observed in both total CD8+ T cells and EBV- or HCMV-specific CD8+ T cells: CD27+CD28+, CD27+CD28−, and CD27−CD28−. A significant number of the CD27−CD28+ subset was observed in total CD8 T cells. However, this subset was barely detectable in EBV- or HCMV-specific CD8+ T cells. Analysis of perforin expression and cytotoxic activity in the first three subsets suggested the following differentiation pathway: CD27+CD28+CD45RA−→CD27+CD28−CD45RA−→CD27−CD28−CD45RA−. This was supported by the observation that the frequency of CCR5+ cells and CCR7+ cells decreased during this sequence. Analysis of CCR5 and CCR7 expression in the CD27+CD28+ memory cell subset demonstrated the presence of three CCR5/CCR7 populations: CCR5−CCR7+, CCR5+CCR7+, and CCR5+CCR7−. These findings suggested the following differentiation pathway: CD27+CD28+CD45RA− (CCR5−CCR7+→CCR5+CCR7+→CCR5+CCR7−)→CD27+CD28−CD45RA−→CD27−CD28−CD45RA−. The presence of a CD27−CD28+ subset with a CCR5+CCR7− phenotype implies a specialized role for this subset in the differentiation of CD8+ T cells.
Both CD4+CD25+ and CD4+CD25− Regulatory Cells Mediate Dominant Transplantation Tolerance
Authors: Graca Luis , Thompson Sara , Lin Chun-Yen , Adams Elizabeth , Cobbold Stephen P. , Waldmann Herman
Published: April 2014

CD4+CD25+ T cells have been proposed as the principal regulators of both self-tolerance and transplantation tolerance. Although CD4+CD25+ T cells do have a suppressive role in transplantation tolerance, so do CD4+CD25− T cells, although 10-fold less potent. Abs to CTLA-4, CD25, IL-10, and IL-4 were unable to abrogate suppression mediated by tolerant spleen cells so excluding any of these molecules as critical agents of suppression. CD4+CD25+ T cells from naive mice can also prevent rejection despite the lack of any previous experience of donor alloantigens. However, this requires many more naive than tolerized cells to provide the same degree of suppression. This suggests that a capacity to regulate transplant rejection pre-exists in naive mice, and may be amplified in “tolerized” mice. Serial analysis of gene expression confirmed that cells sorted into CD4+CD25+ and CD4+CD25− populations were distinct in that they responded to TCR ligation with very different programs of gene expression. Further characterization of the differentially expressed genes may lead to the development of diagnostic tests to monitor the tolerant state.
The Novel IgD Binding Protein fromMoraxella catarrhalisInduces Human B Lymphocyte Activation and Ig Secretion in the Presence of Th2 Cytokines
Authors: Wingren Anette Gjörloff , Hadzic Radinka , Forsgren Arne , Riesbeck Kristian
Published: April 2014

Moraxella IgD binding protein (MID) is a novel bacterial outer membrane protein with IgD-binding properties. MID was purified from the respiratory pathogen Moraxella catarrhalis and is here shown to have B cell stimulatory properties. Purified MID in the range of 0.01–0.1 μg/ml was optimal to induce a proliferative response in human PBL. MID coupled to Sepharose and formalin-fixed M. catarrhalis preparations induced similar proliferative responses in PBL cultures. MID or MID-Sepharose stimulated purified human peripheral B cells as measured by proliferation. In contrast, MID or MID-Sepharose did not activate T cells. Preincubation of purified B cells with anti-IgD Abs inhibited MID-Sepharose-induced B cell proliferation. The addition of IL-4 specifically induced IL-6 production in MID-Sepharose-activated B cells. IgM secretion was detected in B cell cultures stimulated with MID or MID-Sepharose and IL-2 for 10 days. Secretion of IgG and IgA was efficiently induced in cultures from purified B cells stimulated with the combination of MID or MID-Sepharose and IL-4, IL-10, and soluble CD40 ligand, suggesting that Th2-derived cytokines were required for optimal plasma cell generation. Taken together, MID has properties that make it an important tool to study IgD-targeted activation of B cells.
Direct Real-Time Observation of E- and P-Selectin-Mediated Rolling on Cutaneous Lymphocyte-Associated Antigen Immobilized on Western Blots
Authors: Fuhlbrigge Robert C. , King Sandra L. , Dimitroff Charles J. , Kupper Thomas S. , Sackstein Robert
Published: April 2014

Human memory T cells associated with cutaneous inflammatory responses are characterized by their expression of cutaneous lymphocyte-associated Ag (CLA), a carbohydrate determinant differentially expressed on P-selectin glycoprotein ligand-1 (PSGL-1). Although expression of the CLA epitope on PSGL-1 (CLA+ PSGL-1) by memory T cells is associated with acquisition of E-selectin ligand activity, it is not known whether CLA+ PSGL-1, itself, is a ligand for E-selectin on human T cells or whether other glycoproteins, with or without CLA modification, support E-selectin-dependent rolling in shear flow. To address this issue, we developed a method for real-time analysis of functional adhesive interactions between selectin-bearing cells in shear flow with leukocyte ligands resolved by SDS-PAGE and immobilized on standard Western blots. The results of these studies provide direct evidence that CLA+ PSGL-1 is a functional ligand for both E- and P-selectin, confirm that the P-selectin ligand activity of PSGL-1 is independent of CLA modification, and identify a distinct, non-PSGL-1 E-selectin ligand on CLA-positive human memory T cells.
A Three-Megabase Yeast Artificial Chromosome Contig Spanning the C57BL Mouse Igh Locus
Authors: Chevillard Christophe , Ozaki Jennifer , Herring Christopher D. , Riblet Roy
Published: April 2014

The mouse Ig H chain (Igh) complex locus is composed of >100 gene segments encoding the variable, diversity, joining, and constant portions of the Ab H chain protein. To advance the characterization of this locus and to identify all the VH genes, we have isolated the entire region from C57BL/6 and C57BL/10 as a yeast artificial chromosome contig. The mouse Igh locus extends approximately three megabases and contains at least 134 VH genes classified in 15 partially interspersed families. Two non-Igh pseudogenes (Odc-rs8 and Rpl32-rs14) were localized in the distal part of the locus. This physical yeast artificial chromosome map will provide important structure and guidance for the sequencing of this large, complex, and highly repetitive locus.
Oxidant-Mediated Increases in Redox Factor-1 Nuclear Protein and Activator Protein-1 DNA Binding in Asbestos-Treated Macrophages
Authors: Flaherty Dawn M. , Monick Martha M. , Carter A. Brent , Peterson Michael W. , Hunninghake Gary W.
Published: April 2014

Alveolar macrophages have been implicated in the pathogenesis of a number of acute and chronic lung disorders. We have previously shown that normal human alveolar macrophages exhibit decreased DNA binding activity of the transcription factor, AP-1, compared with monocytes. Furthermore, this decrease in AP-1 DNA binding appears to be due to a decrease in the redox active protein, redox factor (Ref)-1. Ref-1 is an important redox regulator of a number of transcription factors, including NF-κB and AP-1. In this study we evaluated the role of asbestos, a prototypic model of chronic fibrotic lung disease, in Ref-1 expression and activity. We found that incubation with low concentrations of crocidolite asbestos (0.5–1.25 μg/cm2) resulted in an increase in nuclear Ref-1 protein after 5 min, with a persistent elevation in protein up to 24 h. Additionally, an increase in nuclear Ref-1 could be induced by treating the cells with an oxidant-generating stimulus (iron loading plus PMA) and inhibited by diphenyleneiodonium chloride, an inhibitor of NADPH oxidase. The asbestos-induced accumulation of nuclear Ref-1 was associated with an increase in AP-1 DNA binding activity. These findings suggest that an exposure associated with fibrotic lung disease, i.e., asbestos, modulates accumulation of nuclear Ref-1 in macrophages, and that this effect is mediated by an oxidant stimulus.
Phospholipases D1 and D2 Regulate Different Phases of Exocytosis in Mast Cells
Authors: Choi Wahn Soo , Kim Young Mi , Combs Christian , Frohman Michael A. , Beaven Michael A.
Published: April 2014

The rat mast cell line RBL-2H3 contains both phospholipase D (PLD)1 and PLD2. Previous studies with this cell line indicated that expressed PLD1 and PLD2 are both strongly activated by stimulants of secretion. We now show by use of PLDs tagged with enhanced green fluorescent protein that PLD1, which is largely associated with secretory granules, redistributes to the plasma membrane in stimulated cells by processes reminiscent of exocytosis and fusion of granules with the plasma membrane. These processes and secretion of granules are suppressed by expression of a catalytically inactive mutant of PLD1 or by the presence of 50 mM 1-butanol but not tert-butanol, an indication that these events are dependent on the catalytic activity of PLD1. Of note, cholera toxin induces translocation of PLD1-labeled granules to the plasma membrane but not fusion of granules with plasma membrane or secretion. Subsequent stimulation of calcium influx with Ag or thapsigargin leads to rapid redistribution of PLD1 to the plasma membrane and accelerated secretion. Also of note, PLD1 is recycled from plasma membrane back to granules within 4 h of stimulation. PLD2, in contrast, is largely confined to the plasma membrane, but it too participates in the secretory process, because expression of catalytically inactive PLD2 also blocks secretion. These data indicate a two-step process: translocation of granules to the cell periphery, regulated by granule-associated PLD1, and a calcium-dependent fusion of granules with the plasma membrane, regulated by plasma membrane-associated PLD2 and possibly PLD1.
In Vivo Generation of Cytotoxic T Cells from Epitopes Displayed on Peptide-Based Delivery Vehicles
Authors: Kawamura Kim S. , Su Ruey-Chyi , Nguyen Linh T. , Elford Alisha R. , Ohashi Pamela S. , Gariépy Jean
Published: April 2014

The development of nonviral, peptide-based constructs able to elicit protective in vivo CTL responses represents a major challenge in the design of future vaccines. We report the design of branched peptide delivery vehicles, termed loligomers, that facilitate the import, processing, and presentation of CTL epitopes onto nascent MHC class I molecules. These complexes are then effectively displayed on the surface of APCs. The intracellular delivery of CTL epitopes by loligomers prolonged the expression of Ag-MHC class I complexes on the surface of APCs in comparison with free CTL epitope alone. Furthermore, the injection of CTL epitope-containing loligomers into mice led to the generation of in vivo CTL responses and the induction of autoimmune disease in an animal model. Synthetic epitope-carrying, peptide-based delivery vehicles may represent useful components to be included in the formulation of future vaccines.
Matrix Fibronectin Increases HIV Stability and Infectivity
Authors: Greco Giampaolo , Pal Sampa , Pasqualini Renata , Schnapp Lynn M.
Published: April 2014

HIV particles are detected extracellularly in lymphoid tissues, a major reservoir of the virus. We previously reported that a polymerized form of fibronectin (FN), superfibronectin (sFN), as well as a fragment of FN, III1-C, enhanced infection of primary CD4+ T cells by HIV-1IIIB. We now show that sFN enhances infection of primary CD4+ T cells by both R5 and X4 strains of HIV-1. Using HIV pseudotyped with different envelope glycoproteins (gp120) and HOS cells transfected with various chemokine receptors alone or in combination with the CD4 molecule, we show that sFN-mediated enhancement requires the CD4 receptor and does not alter the specificity of gp120 for different chemokine receptors. Because the III1-C fragment also resulted in enhancement, we asked whether proteolysis of FN generated fragments capable of enhancing HIV infection. We found that progressive proteolysis of FN by chymotrypsin correlates with an enhancement of HIV infection in both primary CD4+ T cells and the IG5 reporter cell line. Furthermore, incubation of HIV with sFN significantly prolonged infectivity at 37°C compared with dimeric FN or BSA. In conclusion, these results indicate that polymerized (matrix) or degraded (inflammation-associated), but not dimeric (plasma), FN are capable of enhancing infection by HIV-1, independent of the coreceptor specificity of the strains. Moreover, virions bound to matrix FN maintain infectivity for longer periods of time than do virions in suspension. This study suggests that matrix proteins and their conformational status may play a role in the pathogenesis of HIV.
The Role of IL-12 in Maintaining Resistance toLeishmania major
Authors: Park Audrey Y. , Hondowicz Brian , Kopf Manfred , Scott Phillip
Published: April 2014

IL-12p40 is required for the maintenance of resistance during Leishmania major infection. In this study, we addressed how IL-12 mediates this function. First, we demonstrated that both subunits of IL-12, p40 and p35, were required for continued resistance to L. major. Second, using IL-12, IL-4 doubly deficient mice, we investigated the possibility that IL-12 inhibits IL-4-induced outgrowth of Th2 cells that might compete with Th1 cells. We found that even in the absence of a Th2 response, IL-12 was still required to maintain resistance. Next, using adoptive transfer of Thy-1 disparate CD4+ T cells from L. major-healed mice, we were able to show that the loss of a protective response in L. major-infected IL-12-deficient mice is linked with the loss of Th1 cells. In contrast, there was an equal recovery of CD4+ Th1 cells from wild-type and IL-12-deficient mice when transferred into mice that were not challenged with L. major. The ability of Th1 cells to survive regardless of IL-12 levels in the absence of Ag stimulation was confirmed by adoptive transfer studies of CD4+ Th1 cells from DO11.10 TCR transgenic mice. Taken together, these results indicate that, rather than modulating Th2 responses or optimizing IFN-γ production, the critical role for IL-12 in maintaining cell-mediated immunity may be to prevent the loss of Th1 cells during a challenge infection.
C3 Adsorbed to a Polymer Surface Can Form an Initiating Alternative Pathway Convertase
Authors: Andersson Jonas , Ekdahl Kristina Nilsson , Larsson Rolf , Nilsson Ulf R. , Nilsson Bo
Published: April 2014

Contact between blood and a biomaterial surface induces an immediate complement-mediated inflammatory response. Under these conditions, the alternative pathway of complement is often initiated and amplified on the biomaterial surface. Adsorption of a protein such as C3 to a polymer surface induces conformational changes in the protein. Based on the expression on adsorbed C3 of conformational neoepitopes specific for bound C3 fragments, we have hypothesized that adsorbed C3 is able to bind factor B and form a functional C3,Bb convertase. Using a quartz crystal microbalance to monitor binding of proteins to a polymer surface, we have demonstrated that a functional C3-containing alternative pathway convertase can be formed, in particular, in the presence of properdin. These data indicate that adsorption of C3 induces conformational changes that turn C3 into a C3b-like molecule that is able to participate in the functioning of the alternative convertase, and they suggest a new mechanism for complement activation on a biomaterial surface.
Experimental Allergic Encephalomyelitis Is Inhibited in Transgenic Mice Expressing Human C-Reactive Protein
Authors: Szalai Alexander J. , Nataf Serge , Hu Xian-Zhen , Barnum Scott R.
Published: April 2014

We show here using a transgenic model that human C-reactive protein (CRP) protects against experimental allergic encephalomyelitis (EAE) in C57BL/6 mice. In transgenic compared with wild-type females, the duration of the human CRP acute phase response that accompanies the inductive phase of active EAE correlates with a delay in disease onset. In transgenic males, which have higher human CRP expression than females do, EAE is delayed, and its severity is reduced relative to same-sex controls. Furthermore, in male transgenics, there is little or no infiltration of the spinal cord by CD3+ T cells and CD11b+ monocytes and macrophages, and EAE is sometimes prevented altogether. CRP transgenics also resist EAE induced passively by transfer of encephalitogenic T cells from wild-type donors. Human CRP has three effects on cultured encephalitogenic cells that could contribute to the protective effect observed in vivo: 1) CRP inhibits encephalitogenic peptide-induced proliferation of T cells; 2) CRP inhibits production of inflammatory cytokines (TNF-α, IFN-γ) and chemokines (macrophage-inflammatory protein-1α, RANTES, monocyte chemoattractant protein-1); and 3) CRP increases IL-10 production. All three of these actions are realized in vitro only in the presence of high concentrations of human CRP. The combined data suggest that during the acute phase of inflammation accompanying EAE, the high level of circulating human CRP that is achieved in CRP-transgenic mice inhibits the damaging action of inflammatory cells and/or T cells that otherwise support onset and development of EAE.
Possible Involvement of the Vascular Endothelial Growth Factor-Flt-1-Focal Adhesion Kinase Pathway in Chemotaxis and the Cell Proliferation of Osteoclast Precursor Cells in Arthritic Joints
Authors: Matsumoto Yoshihiro , Tanaka Kazuhiro , Hirata Go , Hanada Masuo , Matsuda Shuichi , Shuto Toshihide , Iwamoto Yukihide
Published: April 2014

Vascular endothelial growth factor (VEGF) plays a crucial role in the pathogenesis of inflammatory joint disease, including angiogenesis and synovitis. Rheumatoid arthritis is a chronic inflammatory disease characterized by progressive synovitis and subsequent bone destruction mediated by osteoclasts (OCs). In this study, we investigate the effects of VEGF on OC precursor cells (pOCs) using Raw cells and adjuvant-induced arthritis in rats. OCs and pOCs in the arthritic joints express VEGF and VEGF receptor type I (Flt-1). Raw cells also express Flt-1, and VEGF treatment stimulated chemotaxis, cell proliferation, the association of Flt-1 with focal adhesion kinase (FAK), and the tyrosine phosphorylation of FAK in Raw cells. The tyrosine phosphorylation of FAK was also observed in pOCs in the arthritic joints of adjuvant-induced arthritis. Adenovirus-mediated expression of FAK-related nonkinase in Raw cells inhibited the effects of VEGF in a dominant negative manner. Furthermore, intra-articular injection of the FAK-related nonkinase virus suppressed the recruitment of pOCs and bone destruction. Our results suggest the possible involvement of the VEGF-Flt-1-FAK pathway in inflammatory disease-induced joint destruction.
Exuberant Inflammation in Nicotinamide Adenine Dinucleotide Phosphate-Oxidase-Deficient Mice After Allogeneic Marrow Transplantation
Authors: Yang Shuxia , Panoskaltsis-Mortari Angela , Shukla Mayank , Blazar Bruce R. , Haddad Imad Y.
Published: April 2014

We have shown that NO and superoxide ()contribute to donor T cell-dependent lung dysfunction after bone marrow transplantation (BMT) in mice. We hypothesized that inhibiting production during inducible NO synthase induction would suppress oxidative/nitrative stress and result in less severe lung injury. Irradiated mice lacking the phagocytic NADPH-oxidase (phox−/−), a contributor to generation, were conditioned with cyclophosphamide and given donor bone marrow in the presence or absence of inflammation-inducing allogeneic spleen T cells. On day 7 after allogeneic BMT, survival, weight loss, and indices of lung injury between phox−/− and wild-type mice were not different. However, the majority of macrophages/monocytes from phox−/− mice given donor T cells produced fewer oxidants and contained less nitrotyrosine than cells obtained from T cell-recipient wild-type mice. Importantly, suppressed oxidative stress was associated with marked infiltration of the lungs with inflammatory cells and was accompanied by increased bronchoalveolar lavage fluid levels of the chemoattractants monocyte chemoattractant protein-1 and macrophage-inflammatory protein-1α and impaired clearance of recombinant mouse macrophage-inflammatory protein-1β from the circulation. Furthermore, cultured macrophages/monocytes from NADPH-deficient mice produced 3-fold more TNF-α compared with equal number of cells from NADPH-sufficient mice. The high NO production was not modified during NADPH-oxidase deficiency. We conclude that phox−/− mice exhibit enhanced pulmonary influx of inflammatory cells after BMT. Although NO may contribute to increased production of TNF-α in phox−/− mice, the data suggest that NADPH-oxidase-derived oxidants have a role in limiting inflammation and preventing lung cellular infiltration after allogeneic transplantation.
Plasmid DNA Encoding IFN-γ-Inducible Protein 10 Redirects Antigen-Specific T Cell Polarization and Suppresses Experimental Autoimmune Encephalomyelitis
Authors: Wildbaum Gizi , Netzer Nir , Karin Nathan
Published: April 2014

IFN-γ-inducible protein 10 (IP-10) is a CXC chemokine that stimulates the directional migration of activated T cells, particularly Th1 cells. We demonstrate in this work that during activation this chemokine drives naive CD4+ T cells into Th1 polarization. Administration of plasmid DNA encoding self IP-10 was found capable of breaking down immunological tolerance to IP-10, resulting in the generation of self-specific immunity to the gene product of the vaccine. Despite the CpG motif that drives T cells into Th1, the vaccine redirected the polarization of myelin basic protein-specific T cells into Th2 and conferred the vaccinated recipients a high state of resistance against experimental autoimmune encephalomyelitis, a T cell-mediated autoimmune disease of the CNS. The vaccine also suppressed full-blown ongoing disease in a mouse model of multiple sclerosis. Self-specific Ab to IP-10 developed in protected animals could inhibit leukocyte migration, alter the in vitro Th1/Th2 balance of autoimmune T cells, and adoptively transfer disease suppression. This demonstrates not only the pivotal role of a chemokine in T cell polarization and function but also its potential implications for plasmid DNA gene therapy.
Enhanced Iodination of Thyroglobulin Facilitates Processing and Presentation of a Cryptic Pathogenic Peptide
Authors: Dai Yang D. , Rao Varada P. , Carayanniotis George
Published: April 2014

Increased iodine intake has been associated with the development of experimental autoimmune thyroiditis (EAT), but the biological basis for this association remains poorly understood. One hypothesis has been that enhanced incorporation of iodine in thyroglobulin (Tg) promotes the generation of pathogenic T cell determinants. In this study we sought to test this by using the pathogenic nondominant As-binding Tg peptides p2495 and p2694 as model Ags. SJL mice challenged with highly iodinated Tg (I-Tg) developed EAT of higher severity than Tg-primed controls, and lymph node cells (LNC) from I-Tg-primed hosts showed a higher proliferation in response to I-Tg in vitro than Tg-primed LNC reacting to Tg. Interestingly, I-Tg-primed LNC proliferated strongly in vitro against p2495, but not p2694, indicating efficient and selective priming with p2495 following processing of I-Tg in vivo. Tg-primed LNC did not respond to either peptide. Similarly, the p2495-specific, IL-2-secreting T cell hybridoma clone 5E8 was activated when I-Tg-pulsed, but not Tg-pulsed, splenocytes were used as APC, whereas the p2694-specific T cell hybridoma clone 6E10 remained unresponsive to splenic APC pulsed with Tg or I-Tg. The selective in vitro generation of p2495 was observed in macrophages or dendritic cells, but not in B cells, suggesting differential processing of I-Tg among various APC. These data demonstrate that enhanced iodination of Tg facilitates the selective processing and presentation of a cryptic pathogenic peptide in vivo or in vitro and suggest a mechanism that can at least in part account for the association of high iodine intake and the development of EAT.
Diesel Exposure Favors Th2 Cell Recruitment by Mononuclear Cells and Alveolar Macrophages from Allergic Patients by Differentially Regulating Macrophage-Derived Chemokine and IFN-γ-Induced Protein-10 Production
Authors: Fahy Olivier , Sénéchal Stéphanie , Pène Jérôme , Scherpereel Arnaud , Lassalle Philippe , Tonnel André-Bernard , Yssel Hans , Wallaert Benoı̂t , Tsicopoulos Anne
Published: April 2014

Diesel exhausts and their associated organic compounds may be involved in the recent increase in the prevalence of allergic disorders, through their ability to favor a type 2 immune response. Type 2 T cells have been shown to be preferentially recruited by the chemokines eotaxin (CCL11), macrophage-derived chemokine (MDC, CCL22), and thymus activation-regulated chemokine (CCL17) through their interaction with CCR3 and CCR4, respectively, whereas type 1 T cells are mainly recruited by IFN-γ-induced protein-10 (CXCL10) through CXCR3 binding. The aim of the study was to evaluate the effect of diesel exposure on the expression of chemokines involved in type 1 and 2 T cell recruitment. PBMC and alveolar macrophages from house dust mite allergic patients were incubated with combinations of diesel extracts and Der p 1 allergen, and chemokine production was analyzed. Diesel exposure alone decreased the constitutive IP-10 production, while it further augmented allergen-induced MDC production, resulting in a significantly increased capacity to chemoattract human Th2, but not Th1 clones. Inhibition experiments with anti-type 1 or type 2 cytokine Abs as well as cytokine mRNA kinetic evaluation showed that the chemokine variations were not dependent upon IL-4, IL-13, or IFN-γ expression. In contrast, inhibition of the B7:CD28 pathway using a CTLA-4-Ig fusion protein completely inhibited diesel-dependent increase of allergen-induced MDC production. This inhibition was mainly dependent upon the CD86 pathway and to a lesser extent upon the CD80 pathway. These results suggest that the exposure to diesel exhausts and allergen may likely amplify a deleterious type 2 immune response via a differential regulation of chemokine production through the CD28 pathway.
The Kinetics of In Vivo Priming of CD4 and CD8 T Cells by Dendritic/Tumor Fusion Cells in MUC1-Transgenic Mice
Authors: Koido Shigeo , Tanaka Yasuhiro , Chen Dongshu , Kufe Donald , Gong Jianlin
Published: April 2014

Previous work has demonstrated that dendritic/tumor fusion cells induce potent antitumor immune responses in vivo and in vitro. However, little is known about the migration and homing of fusion cells after s.c. injection or the kinetics of CD4+ and CD8+ T cell activation. In the present study, fluorescence-labeled dendritic/MUC1-positive tumor fusion cells (FC/MUC1) were injected s.c. into MUC1-transgenic mice. The FC/MUC1 migrated to draining lymph nodes and were closely associated with T cells in a pattern comparable with that of unfused dendritic cells. Immunization of MUC1-transgenic mice with FC/MUC1 resulted in proliferation of T cells and induced MUC1-specific CD8+ CTL. Moreover, CD4+ T cells activated by FC/MUC1 were multifunctional effectors that produced IL-2, IFN-γ, IL-4, and IL-10. These findings indicate that both CD4+ and CD8+ T cells can be primed in vivo by FC/MUC1 immunization.
Fucosyltransferase VII-Deficient Mice with Defective E-, P-, and L-Selectin Ligands Show Impaired CD4+ and CD8+ T Cell Migration into the Skin, but Normal Extravasation into Visceral Organs
Authors: Erdmann Iris , Scheidegger E. Paul , Koch Frauke K. , Heinzerling Lucie , Odermatt Bernhard , Burg Günter , Lowe John B. , Kündig Thomas M.
Published: April 2014

The first step of leukocyte extravasation, leukocyte rolling, is mediated by E-, P-, and L-selectins. Mice deficient for α-1,3-fucosyltransferase VII (FucTVII)−/− are characterized by deficiency of E-, P-, and L-selectin ligand activity. This model system was used to evaluate the role of the interactions of selectins with their ligands in T and B cell responses. In the present study, FucTVII−/− mice showed reduced CD4+ T cell-mediated contact hypersensitivity reactions of the ears to FITC as well as reduced CD8+ T cell-mediated delayed-type hypersensitivity reactions of the footpads against lymphocytic choriomeningitis virus infection. As Langerhans cell migration to local lymph nodes as well as CD4+ and CD8+ T cell induction were found to be normal, the afferent arm of these reactions was not impaired. The reduced inflammatory reactions of the skin were due to inefficient lymphocyte extravasation into the skin. In contrast, extravasation of CD4+ and CD8+ T cells into visceral organs, such as the ovaries or the brain, was not impaired in FucTVII−/− mice. Elimination of vaccinia virus and of lymphocytic choriomeningitis virus from ovaries and brain, as well as elimination of tumor cells from several visceral organs was normal. Thus, interactions of selectins with their ligands are important for lymphocyte homing into the skin, but not for lymphocyte extravasation into visceral organs.
Defective Fas Ligand Expression and Activation-Induced Cell Death in the Absence of IL-2-Inducible T Cell Kinase
Authors: Miller Andrew T. , Berg Leslie J.
Published: April 2014

The Tec family tyrosine kinase, IL-2-inducible T cell kinase (Itk), plays an important role in TCR signaling. Studies of T cells from Itk-deficient mice have demonstrated that Itk is critical for the activation of phospholipase-Cγ1, leading to calcium mobilization in response to TCR stimulation. This biochemical defect results in reduced IL-2 production by Itk-deficient T cells. To further characterize the downstream effects of the Itk deficiency, we crossed Itk−/− mice to a TCR-transgenic line and examined T cell responses to stimulation by peptide plus APC. These studies show that Itk is required for maximal activation of early growth responses 2 and 3 and Fas ligand transcription after TCR stimulation. These transcriptional defects lead to reduced activation-induced cell death of stimulated Itk−/− T cells, both in vitro and in vivo. Together these studies define an important role for Itk in TCR signaling, leading to cytokine gene expression and activation-induced cell death.
IL-12 Induces Monocyte IL-18 Binding Protein Expression Via IFN-γ
Authors: Veenstra Korina G. , Jonak Zdenka L. , Trulli Stephen , Gollob Jared A.
Published: April 2014

IL-18 is a Th1 cytokine that synergizes with IL-12 and IL-2 in the stimulation of lymphocyte IFN-γ production. IL-18 binding protein (IL-18BP) is a recently discovered inhibitor of IL-18 that is distinct from the IL-1 and IL-18 receptor families. In this report we show that IL-18BPa, the IL-18BP isoform with the highest affinity for IL-18, was strongly induced by IL-12 in human PBMC. Other Th1 cytokines, including IFN-γ, IL-2, IL-15, and IL-18, were also capable of augmenting IL-18BPa expression. In contrast, IL-1α, IL-1β, TNF-α, IFN-γ-inducible protein-10, and Th2 cytokines such as IL-4 and IL-10 did not induce IL-18BPa. Although monocytes were found to be the primary source of IL-18BPa, the induction of IL-18BPa by IL-12 was mediated through IFN-γ derived predominantly from NK cells. IL-18BPa production was observed in cancer patients receiving recombinant human IL-12 and correlated with the magnitude of IFN-γ production. The IFN-γ/IL-18BPa negative feedback loop identified in this study may be capable of broadly controlling immune activation by cytokines that synergize with IL-18 to induce IFN-γ and probably plays a key role in the modulation of both innate and adaptive immunity.