bluuebunny/crossref_metadata_2025 huggingface dataset:
The Salmonella Pathogenicity Island (SPI)-2 and SPI-1 Type III Secretion Systems Allow Salmonella Serovar typhimurium to Trigger Colitis via MyD88-Dependent and MyD88-Independent Mechanisms
Authors: Hapfelmeier Siegfried , Stecher Bärbel , Barthel Manja , Kremer Marcus , Müller Andreas J. , Heikenwalder Mathias , Stallmach Thomas , Hensel Michael , Pfeffer Klaus , Akira Shizuo , Hardt Wolf-Dietrich
Published: April 2014

Salmonella typhimurium can colonize the gut, invade intestinal tissues, and cause enterocolitis. In vitro studies suggest different mechanisms leading to mucosal inflammation, including 1) direct modulation of proinflammatory signaling by bacterial type III effector proteins and 2) disruption or penetration of the intestinal epithelium so that penetrating bacteria or bacterial products can trigger innate immunity (i.e., TLR signaling). We studied these mechanisms in vivo using streptomycin-pretreated wild-type and knockout mice including MyD88−/− animals lacking an adaptor molecule required for signaling via most TLRs. The Salmonella SPI-1 and the SPI-2 type III secretion systems (TTSS) contributed to inflammation. Mutants that retain only a functional SPI-1 (M556; sseD::aphT) or a SPI-2 TTSS (SB161; ΔinvG) caused attenuated colitis, which reflected distinct aspects of the colitis caused by wild-type S. typhimurium: M556 caused diffuse cecal inflammation that did not require MyD88 signaling. In contrast, SB161 induced focal mucosal inflammation requiring MyD88. M556 but not SB161 was found in intestinal epithelial cells. In the lamina propria, M556 and SB161 appeared to reside in different leukocyte cell populations as indicated by differential CD11c staining. Only the SPI-2-dependent inflammatory pathway required aroA-dependent intracellular growth. Thus, S. typhimurium can use two independent mechanisms to elicit colitis in vivo: SPI-1-dependent and MyD88-independent signaling to epithelial cells and SPI-2-dependent intracellular proliferation in the lamina propria triggering MyD88-dependent innate immune responses.
A Chemokine-Dependent Stromal Induction Mechanism for Aberrant Lymphocyte Accumulation and Compromised Lymphatic Return in Rheumatoid Arthritis
Authors: Burman Angela , Haworth Oliver , Hardie Debbie L. , Amft E. Nicole , Siewert Christiane , Jackson David G. , Salmon Mike , Buckley Christopher D.
Published: April 2014

According to the current model for tissue-specific homing, specificity is conferred by the selective recruitment of lymphocyte populations from peripheral blood, based on their expression of chemokine and adhesion receptors (endothelial selection). In this study, we provide evidence for an alternative stromal induction mechanism that operates in chronic inflammation. We show that the human rheumatoid synovial microenvironment directly induces functional inflammatory (CCR5 and CXCR3) and constitutive (CCR7 and CXCR4) chemokine receptors on infiltrating CD4+ T cells. Expression of the corresponding inflammatory chemokine ligands (CCL5 and CXCL11) was confined to stromal areas in the synovium. However, expression of the constitutive ligands (CCL19 and CXCL12) was inappropriately high on both vascular and lymphatic endothelium, suggesting that the vascular to lymphatic chemokine gradient involved in lymphatic recirculation becomes subverted in the rheumatoid synovium. These results challenge the view that leukocyte trafficking is regulated solely by selective recruitment of pre-existing chemokine receptor-positive cells from peripheral blood, by providing an alternative explanation based on aberrant lymphocyte retention and compromised lymphatic return.
NF-κB Activation Is Not Required for Chlamydia trachomatis Inhibition of Host Epithelial Cell Apoptosis
Authors: Xiao Yangming , Zhong Youmin , Su Heng , Zhou Zhiguang , Chiao Paul , Zhong Guangming
Published: April 2014

Chlamydia trachomatis, an obligate intracellular bacterial species, is known to inhibit host cell apoptosis. However, the chlamydial antiapoptotic mechanism is still not clear. Because NF-κB activation is antiapoptotic, we tested the potential role of NF-κB activation in chlamydial antiapoptotic activity in the current study. First, no obvious NF-κB activation was detected in the chlamydia-infected cells when these cells were resistant to apoptosis induced via either the intrinsic or extrinsic apoptosis pathways. Second, inhibition of NF-κB activation with pharmacologic reagents failed to block the chlamydial antiapoptotic activity. Finally, NF-κB p65 gene deletion did not prevent chlamydia from inhibiting host cell apoptosis. These observations together have demonstrated that NF-κB activation is not required for the chlamydial antiapoptotic activity.
Cathepsin S Is Required for Murine Autoimmune Myasthenia Gravis Pathogenesis
Authors: Yang Huan , Kala Mrinalini , Scott Benjamin G. , Goluszko Elzbieta , Chapman Harold A. , Christadoss Premkumar
Published: April 2014

Because presentation of acetylcholine receptor (AChR) peptides to T cells is critical to the development of myasthenia gravis, we examined the role of cathepsin S (Cat S) in experimental autoimmune myasthenia gravis (EAMG) induced by AChR immunization. Compared with wild type, Cat S null mice were markedly resistant to the development of EAMG, and showed reduced T and B cell responses to AChR. Cat S null mice immunized with immunodominant AChR peptides showed weak responses, indicating failed peptide presentation accounted for autoimmune resistance. A Cat S inhibitor suppressed in vitro IFN-γ production by lymph node cells from AChR-immunized, DR3-bearing transgenic mice. Because Cat S null mice are not severely immunocompromised, Cat S inhibitors could be tested for their therapeutic potential in EAMG.
IFN-γ and IL-10 Mediate Parasite-Specific Immune Responses of Cord Blood Cells Induced by Pregnancy-Associated Plasmodium falciparum Malaria
Authors: Brustoski Kim , Möller Ulrike , Kramer Martin , Petelski Annika , Brenner Stephan , Palmer Dupeh R. , Bongartz Martina , Kremsner Peter G. , Luty Adrian J. F. , Krzych Urszula
Published: April 2014

Available evidence suggests that immune cells from neonates born to mothers with placental Plasmodium falciparum (Pf) infection are sensitized to parasite Ag in utero but have reduced ability to generate protective Th1 responses. In this study, we detected Pf Ag-specific IFN-γ+ T cells in cord blood from human neonates whose mothers had received treatment for malaria or who had active placental Pf infection at delivery, with responses being significantly reduced in the latter group. Active placental malaria at delivery was also associated with reduced expression of monocyte MHC class I and II in vivo and following short term in vitro coculture with Pf Ag compared with levels seen in neonates whose mothers had received treatment during pregnancy. Given that APC activation and Th1 responses are driven in part by IFN-γ and down-regulated by IL-10, we examined the role of these cytokines in modulating the Pf Ag-specific immune responses in cord blood samples. Exogenous recombinant human IFN-γ and neutralizing anti-human IL-10 enhanced T cell IFN-γ production, whereas recombinant human IFN-γ also restored MHC class I and II expression on monocytes from cord blood mononuclear cells cocultured with Pf Ag. Accordingly, active placental malaria at delivery was associated with increased frequencies of Pf Ag-specific IL-10+CD4+ T cells in cord blood mononuclear cell cultures from these neonates. Generation and maintenance of IL-10+ T cells in utero may thus contribute to suppression of APC function and Pf Ag-induced Th1 responses in newborns born to mothers with placental malaria at delivery, which may increase susceptibility to infection later in life.
Cutting Edge: Contact-Mediated Suppression by CD4+CD25+ Regulatory Cells Involves a Granzyme B-Dependent, Perforin-Independent Mechanism
Authors: Gondek David C. , Lu Li-Fan , Quezada Sergio A. , Sakaguchi Shimon , Noelle Randolph J.
Published: April 2014

CD4+CD25+ regulatory T cells (Treg) are potent immunosuppressive cells that are pivotal in the regulation of peripheral tolerance. In this report, we identify granzyme B (GZ-B) as one of the key components of Treg-mediated suppression. Induction of regulatory activity is correlated with the up-regulation of GZ-B expression. Proof of a functional involvement of GZ-B in contact-mediated suppression by Treg is shown by the reduced ability of Treg from GZ-B−/− mice to suppress as efficiently as Treg from WT mice. GZ-B-mediated suppression is perforin independent, because suppression by Treg from perforin−/− and WT is indistinguishable. Additionally, suppression mediated by Treg appears to be mediated, in part, by the induction of apoptosis in the CD4+CD25− effector cell. In summary, GZ-B is one of the key mechanisms through which CD4+CD25+ Treg induce cell contact-mediated suppression.
Intercellular Adhesion Molecule-1/LFA-1 Cross Talk Is a Proximate Mediator Capable of Disrupting Immune Integration and Tolerance Mechanism at the Feto-Maternal Interface in Murine Pregnancies
Authors: Blois Sandra , Tometten Mareike , Kandil Judith , Hagen Evelin , Klapp Burghard F. , Margni Ricardo A. , Arck Petra C.
Published: April 2014

Our understanding why a woman’s immune system does not reject her histoincompatible fetus is still very limited. Distinct insights into the mechanisms involved in pregnancy maintenance may help us to prevent pregnancy complications, e.g., miscarriages or pre-eclampsia. Immune integration and tolerance at the feto-maternal interface appear to be indispensable for successful pregnancy maintenance. Little is known about the cross talk between ICAM-1, expressed on epithelium, endothelium, and APC, and its ligand, LFA-1, at the feto-maternal interface. However, based on the role of ICAM-1/LFA-1 in allograft acceptance or rejection upon transplantation, adhesion molecules are likely to interfere with successful pregnancy outcome. In this study, we tested the hypothesis that ICAM-1/LFA-1 pathways may be involved in pregnancy rejection in murine models. By blocking ICAM-1/LFA-1-mediated intercellular adhesion events, we show that fetal immune acceptance is restored in challenged pregnancies (e.g., upon exposure to sound stress), and adoptive transfer of LFA-1 cells into pregnant mice induces rejection only in abortion-prone mouse models. ICAM-1/LFA-1 cross talk leads to increased recruitment of proinflammatory cells to the implantation site, promotes dendritic cell maturation in the decidua, and subsequently induces additional local Th1 polarization via mature dendritic cells. Furthermore, our observations clearly point out that mechanisms of fetal tolerance, e.g., indoleamine 2,3-dioxygenase expression, presence of CD4+CD25bright regulatory T cells, and synthesis of asymmetric Abs, are ICAM-1/LFA-1 dependent. Hence, our data shed light on a hierarchical network of immune integration at the feto-maternal interface, in which ICAM-1/LFA-1 cross talk is clearly a proximate mediator capable of disrupting successful pregnancy maintenance.
Rapid Activation of Spleen Dendritic Cell Subsets following Lymphocytic Choriomeningitis Virus Infection of Mice: Analysis of the Involvement of Type 1 IFN
Authors: Montoya Maria , Edwards Matthew J. , Reid Delyth M. , Borrow Persephone
Published: April 2014

In this study, we report the dynamic changes in activation and functions that occur in spleen dendritic cell (sDC) subsets following infection of mice with a natural murine pathogen, lymphocytic choriomeningitis virus (LCMV). Within 24 h postinfection (pi), sDCs acquired the ability to stimulate naive LCMV-specific CD8+ T cells ex vivo. Conventional (CD11chigh CD8+ and CD4+) sDC subsets rapidly up-regulated expression of costimulatory molecules and began to produce proinflammatory cytokines. Their tendency to undergo apoptosis ex vivo simultaneously increased, and in vivo the number of conventional DCs in the spleen decreased markedly, dropping ∼2-fold by day 3 pi. Conversely, the number of plasmacytoid (CD11clowB220+) DCs in the spleen increased, so that they constituted almost 40% of sDCs by day 3 pi. Type 1 IFN production was up-regulated in plasmacytoid DCs by 24 h pi. Analysis of DC activation and maturation in mice unable to respond to type 1 IFNs implicated these cytokines in driving infection-associated phenotypic activation of conventional DCs and their enhanced tendency to undergo apoptosis, but also indicated the existence of type 1 IFN-independent pathways for the functional maturation of DCs during LCMV infection.
Induction of Obliterative Airway Disease in Murine Tracheal Allografts by CD8+ CTLs Recognizing a Single Minor Histocompatibility Antigen
Authors: Higuchi Toru , Maruyama Takahiro , Jaramillo Andrés , Mohanakumar Thalachallour
Published: April 2014

The role of minor histocompatibility Ag (mHAg)-specific CD8+ CTLs in the pathogenesis of chronic lung allograft rejection (bronchiolitis obliterans syndrome) remains to be elucidated. Thus, the goal of this study was to define the role of a single mHAg mismatch at the polymorphic H13 allele in the development of obliterative airway disease (OAD) after murine heterotopic tracheal transplantation. The H13a and H13b alleles encode for the SSVVGVWYL (SVL9) and SSVIGVWYL (SIL9) mHAgs, respectively, presented in the context of the H2Db MHC class I molecule. Toward this, C56BL/10SnJ (H13a) tracheal allografts were transplanted into congenic B10.CE-H13b Aw(30NX)/Sn (H13b) recipients. The allografts were harvested at 30, 60, and 90 days after transplantation, and OAD lesions (epithelial damage, cellular infiltration, and luminal fibrosis) were confirmed histologically. Selected groups of mice were immunized (s.c.) or tolerized (i.v.) with the SVL9 peptide before transplantation. This single mHAg mismatch induced the development of OAD within 90 days. SVL9 immunization significantly accelerated the kinetics of the OAD lesions. In contrast, SVL9 tolerization completely abrogated the development of OAD. This was correlated with a complete abrogation of H13a-specific CD8+ CTL responses with a significant reduction in the frequency of IFN-γ-producing CTLs and the activation of TGF-β-producing CD8+ T cells. In conclusion, a single mHAg mismatch can induce the development of OAD. These data also suggest that mHAg-reactive CD8+ CTLs may play an important role in the pathogenesis of chronic lung allograft rejection in humans.
Multiple Intracellular Routes in the Cross-Presentation of a Soluble Protein by Murine Dendritic Cells
Authors: Palliser Deborah , Guillen Eduardo , Ju Mindy , Eisen Herman N.
Published: April 2014

Soluble heat shock fusion proteins (Hsfp) stimulate mice to produce CD8+ CTL, indicating that these proteins are cross-presented by dendritic cells (DC) to naive CD8 T cells. We report that cross-presentation of these proteins depends upon their binding to DC receptors, likely belonging to the scavenger receptor superfamily. Hsfp entered DC by receptor-mediated endocytosis that was either inhibitable by cytochalasin D or not inhibitable, depending upon aggregation state and time. Most endocytosed Hsfp was transported to lysosomes, but not the small cross-presented fraction that exited early from the endocytic pathway and required access to proteasomes and TAP. Naive CD8 T cell (2C and OT-I) responses to DC incubated with Hsfp at 1 μM were matched by incubating DC with cognate octapeptides at 1–10 pM, indicating that display of very few class I MHC-peptide complexes per DC can be sufficient for cross-presentation. With an Hsfp (heat shock protein-OVA) having peptide sequences for both CD4+ (OT-II) and CD8+ (OT-I) cells, the CD4 cells responded far more vigorously than the CD8 cells and many more class II MHC-peptide than class I MHC-peptide complexes were displayed.
Contrasting Effects of Low-Dose IL-2 on Vaccine-Boosted Simian Immunodeficiency Virus (SIV)-Specific CD4+ and CD8+ T Cells in Macaques Chronically Infected with SIVmac251
Authors: Nacsa Janos , Edghill-Smith Yvette , Tsai Wen-Po , Venzon David , Tryniszewska Elzbieta , Hryniewicz Anna , Moniuszko Marcin , Kinter Audrey , Smith Kendall A. , Franchini Genoveffa
Published: April 2014

IL-2, the first cytokine discovered with T cell growth factor activity, is now known to have pleiotropic effects on T cells. For example, it can promote growth, survival, and differentiation of Ag-selected cells, or facilitate Ag-induced cell death of T cells when Ag persists, and in vivo, it is thought to contribute to the regulation of the size of adaptive T cell response. IL-2 is deficient in HIV-1 infection and has been used in the management of HIV-1-infected individuals undergoing antiretroviral therapy. In this study, we investigated how continuous low-dose IL-2 affected the CD4+ and CD8+ T cell response induced by two inoculations of a canarypox recombinant SIV-based vaccine candidate in healthy macaques chronically infected with SIVmac251. These macaques had normal levels of CD4+ T cells at the beginning of antiretroviral therapy treatment. Vaccination in the presence of IL-2 significantly augmented Gag-specific CD8+ T cell responses, but actually reduced Gag-specific CD4+ T cell responses. Although IL-2 at low doses did not change the overall concentration of circulating CD4+ or CD8+ T cells, it expanded the frequency of CD4+CD25+ T cells. Depletion of the CD4+CD25+ T cells in vitro, however, did not result in a reconstitution of Gag-specific CD4+ responses or augmentation of SIV-specific CD8+ T cell responses. Thus, we conclude that the decrease in virus-specific CD4+ T cell response may be due to IL-2-promoted redistribution of cells from the circulation, or due to Ag-induced cell death, rather than suppression by a T regulatory population.
Engagement of NKG2D by Cognate Ligand or Antibody Alone Is Insufficient to Mediate Costimulation of Human and Mouse CD8+ T Cells
Authors: Ehrlich Lauren I. Richie , Ogasawara Kouetsu , Hamerman Jessica A. , Takaki Rayna , Zingoni Alessandra , Allison James P. , Lanier Lewis L.
Published: April 2014

CD8+ T cells require a signal through a costimulatory receptor in addition to TCR engagement to become activated. The role of CD28 in costimulating T cell activation is well established. NKG2D, a receptor found on NK cells, CD8+ αβ-TCR+ T cells, and γδ-TCR+ T cells, has also been implicated in T cell costimulation. In this study we have evaluated the role of NKG2D in costimulating mouse and human naive and effector CD8+ T cells. Unexpectedly, in contrast to CD28, NKG2D engagement by ligand or mAb is not sufficient to costimulate naive or effector CD8+ T cell responses in conventional T cell populations. While NKG2D did not costimulate CD8+ T cells on its own, it was able to modify CD28-mediated costimulation of human CD8+ T cells under certain contitions. It is, therefore, likely that NKG2D acts as a costimulatory molecule only under restricted conditions or requires additional cofactors.
Vascular Endothelial Cells Have Impaired Capacity to Present Immunodominant, Antigenic Peptides: A Mechanism of Cell Type-Specific Immune Escape
Authors: Kummer Marco , Lev Avital , Reiter Yoram , Biedermann Barbara C.
Published: April 2014

Vascular endothelial cells (EC) are an exposed target tissue in the course of CTL-mediated alloimmune diseases such as graft-vs-host disease (GVHD) or solid organ transplant rejection. The outcome of an interaction between CTL and target cells is determined by the amount of Ag presented and the costimulatory signals delivered by the target cells. We compared human EC with leukocytes and epithelial cells as targets for peptide-specific, MHC class I-restricted CTL clones. EC were poor targets for immunodominant CTL. Both endogenously processed antigenic proteins and exogenously added antigenic peptides are presented at 50- to 5000-fold lower levels on EC compared with any other target cell analyzed. This quantitative difference fully explained the poor CTL-mediated killing of EC. There was no evidence that lack of costimulation would contribute significantly to this cell type-specific difference in CTL activation. An HLA-A2-specific CTL clone that killed a broad selection of HLA A2-positive target cells equally well, killed EC less efficiently. Our data suggest that EC present a different Ag repertoire compared with other cell types. By this mechanism, these cells may escape an attack by effector CTL, which have been educated by professional APCs and are specific for immunodominant antigenic peptides.
CCR8 Is Expressed by Antigen-Elicited, IL-10-Producing CD4+CD25+ T Cells, Which Regulate Th2-Mediated Granuloma Formation in Mice
Authors: Freeman Christine M. , Chiu Bo-Chin , Stolberg Valerie R. , Hu Jerry , Zeibecoglou Kyriaki , Lukacs Nicholas W. , Lira Sergio A. , Kunkel Steven L. , Chensue Stephen W.
Published: April 2014

CCR8 was initially described as a Th2 cell-restricted receptor, but this has not been fully tested in vivo. The present study used ex vivo and in vivo approaches to examine the distribution and functional significance of CCR8 among CD4+ T cells. Populations of cytokine-secreting CD4+ T cells were generated in primed mice with Th1 or Th2 cell-mediated pulmonary granulomas, respectively elicited by i.v. challenge with either Mycobacteria bovis purified protein derivative- or Schistosoma mansoni egg Ag (SEA)-coated beads. Cytokine-producing CD4+ T cells were isolated from Ag-stimulated draining lymph node cultures by positive selection. Quantitative analysis of cytokine mRNA indicated enriched populations of IFN-γ-, IL-4-, and IL-10-producing cells. Analysis of chemokine receptor mRNA indicated that IL-10+ cells selectively expressed CCR8 in the SEA bead-elicited type 2 response. The IL-10+CCR8+ populations were CD25+ and CD44+ but lacked enhanced Foxp3 expression. Adoptive transfer to naive recipients indicated that IL-10+ T cells alone could not transfer type 2 inflammation. Analysis of SEA bead-challenged CCR8−/− mice indicated significantly impaired IL-10 production as well as reductions in granuloma eosinophils. Adoptive transfer of CD4+CCR8+/+ T cells corrected cytokine and inflammation defects, but the granuloma eosinophil recruitment defect persisted when donor cells were depleted of IL-10+ cells. Accordingly, local IL-10 production correlated with CCR8 ligand (CCL1) expression and the appearance of CCR8+ cells in granulomatous lungs. Thus, IL-10-producing, CCR8+CD4+CD25+CD44+ T cells are generated during SEA challenge, which augment the Th2-mediated eosinophil-rich response to the parasite Ags.
ICOS-Mediated Costimulation on Th2 Differentiation Is Achieved by the Enhancement of IL-4 Receptor-Mediated Signaling
Authors: Watanabe Masashi , Watanabe Shiho , Hara Yasushi , Harada Yohsuke , Kubo Masato , Tanabe Kazunari , Toma Hiroshi , Abe Ryo
Published: April 2014

ICOS is the third member of the CD28 family molecules and plays a critical role in many T cell-dependent immune responses. Although accumulated data suggest that ICOS costimulatory signals play an important role in Th2-mediated immune responses, the molecular basis for this selective differentiation mechanism is largely unknown. To clarify this mechanism, we used DO11.10 TCR transgenic ICOS−/− mice and evaluated the nature of ICOS costimulatory signals during the process of Ag-specific activation and differentiation of naive CD4+ T cells. Results obtained from these experiments demonstrated that Ag stimulation of naive CD4+ T cells in the absence of an ICOS signal resulted in impaired Th2 development. Unlike previous reports, we found that primary IL-4 production by these T cells was intact and that IL-4R sensitivity of these T cells was reduced as evidenced by a profound defect in IL-4-induced Stat6 phosphorylation and the early induction of GATA-3. The fact that ICOS ligation of wild-type T cells significantly enhanced IL-4-induced Stat6 phosphorylation and primary GATA-3 induction, but not IL-4 transcription, of naive CD4+ T cells was consistent with the results obtained from ICOS−/− T cell experiments. These observations led us to propose that the predominant effect of ICOS-mediated costimulation on Th2 differentiation is achieved by the enhancement of IL-4R-mediated signaling.
Negative Regulation of Phagocytosis in Macrophages by the CD47-SHPS-1 System
Authors: Okazawa Hideki , Motegi Sei-ichiro , Ohyama Naoko , Ohnishi Hiroshi , Tomizawa Takeshi , Kaneko Yoriaki , Oldenborg Per-Arne , Ishikawa Osamu , Matozaki Takashi
Published: April 2014

Src homology 2 domain-containing protein tyrosine phosphatase (SHP) substrate-1 (SHPS-1) is a transmembrane protein that is expressed predominantly in macrophages. Its extracellular region interacts with the transmembrane ligand CD47 expressed on the surface of adjacent cells, and its cytoplasmic region binds the protein tyrosine phosphatases SHP-1 and SHP-2. Phagocytosis of IgG- or complement-opsonized RBCs by peritoneal macrophages derived from mice that express a mutant SHPS-1 protein that lacks most of the cytoplasmic region was markedly enhanced compared with that apparent with wild-type macrophages. This effect was not observed either with CD47-deficient RBCs as the phagocytic target or in the presence of blocking Abs to SHPS-1. Depletion of SHPS-1 from wild-type macrophages by RNA interference also promoted FcγR-mediated phagocytosis of wild-type RBCs. Ligation of SHPS-1 on macrophages by CD47 on RBCs promoted tyrosine phosphorylation of SHPS-1 and its association with SHP-1, whereas tyrosine phosphorylation of SHPS-1 was markedly reduced in response to cross-linking of FcγRs. Treatment with inhibitors of PI3K or of Syk, but not with those of MEK or Src family kinases, abolished the enhancement of FcγR-mediated phagocytosis apparent in macrophages from SHPS-1 mutant mice. In contrast, FcγR-mediated tyrosine phosphorylation of Syk, Cbl, or the γ subunit of FcR was similar in macrophages from wild-type and SHPS-1 mutant mice. These results suggest that ligation of SHPS-1 on macrophages by CD47 promotes the tyrosine phosphorylation of SHPS-1 and thereby prevents the FcγR-mediated disruption of the SHPS-1-SHP-1 complex, resulting in inhibition of phagocytosis. The inhibition of phagocytosis by the SHPS-1-SHP-1 complex may be mediated at the level of Syk or PI3K signaling.
Dual Requirement for the Igα Immunoreceptor Tyrosine-Based Activation Motif (ITAM) and a Conserved Non-Igα ITAM Tyrosine in Supporting Igαβ-Mediated B Cell Development
Authors: Pike Kelly A. , Ratcliffe Michael J. H.
Published: April 2014

Surface Ig (sIg) expression is a critical checkpoint during avian B cell development. Only cells that express sIg colonize bursal follicles, clonally expand, and undergo Ig diversification by gene conversion. Expression of a heterodimer, in which the extracellular and transmembrane domains of murine CD8α or CD8β are fused to the cytoplasmic domains of chicken Igα (chIgα) or Igβ, respectively (murine CD8α (mCD8α):chIgα + mCD8β:chIgβ), or an mCD8α:chIgα homodimer supported bursal B cell development as efficiently as endogenous sIg. In this study we demonstrate that B cell development, in the absence of chIgβ, requires both the Igα ITAM and a conserved non-ITAM Igα tyrosine (Y3) that has been associated with binding to B cell linker protein (BLNK). When associated with the cytoplasmic domain of Igβ, the Igα ITAM is not required for the induction of strong calcium mobilization or BLNK phosphorylation, but is still necessary to support B cell development. In contrast, mutation of the Igα Y3 severely compromised calcium mobilization when expressed as either a homodimer or a heterodimer with the cytoplasmic domain of Igβ. However, coexpression of the cytoplasmic domain of Igβ partially complemented the Igα Y3 mutation, rescuing higher levels of BLNK phosphorylation and, more strikingly, supporting B cell development.
Progressive Differentiation and Commitment of CD8+ T Cells to a Poorly Cytolytic CD8low Phenotype in the Presence of IL-4
Authors: Kienzle Norbert , Olver Stuart , Buttigieg Kathy , Groves Penny , Janas Michelle L. , Baz Adriana , Kelso Anne
Published: April 2014

Exposure to IL-4 during activation of naive murine CD8+ T cells leads to generation of IL-4-producing effector cells with reduced surface CD8, low perforin, granzyme B and granzyme C mRNA, and poor cytolytic function. We show in this study that maximal development of these cells depended on exposure to IL-4 for the first 5 days of activation. Although IL-4 was not required at later times, CD8 T cell clones continued to lose surface CD8 expression with prolonged culture, suggesting commitment to the CD8low phenotype. This state was reversible in early differentiation. When single CD8low cells from 4-day cultures were cultured without IL-4, 65% gave rise to clones that partly or wholly comprised CD8high cells; the proportion of reverted clones was reduced or increased when the cells were cloned in the presence of IL-4 or anti-IL-4 Ab, respectively. CD8 expression positively correlated with perforin and granzyme A, B, and C mRNA, and negatively correlated with IL-4 mRNA levels among these clones. By contrast, most CD8low cells isolated at later time points maintained their phenotype, produced IL-4, and exhibited poor cytolytic function after many weeks in the absence of exogenous IL-4. We conclude that IL-4-dependent down-regulation of CD8 is associated with progressive differentiation and commitment to yield IL-4-producing cells with little cytolytic activity. These data suggest that the CD4−CD8− cells identified in some disease states may be the product of a previously unrecognized pathway of effector differentiation from conventional CD8+ T cells.
Low TLR4 Expression by Liver Dendritic Cells Correlates with Reduced Capacity to Activate Allogeneic T Cells in Response to Endotoxin
Authors: De Creus An , Abe Masanori , Lau Audrey H. , Hackstein Holger , Raimondi Giorgio , Thomson Angus W.
Published: April 2014

Signaling via TLRs results in dendritic cell (DC) activation/maturation and plays a critical role in the outcome of primary immune responses. So far, no data exist concerning TLR expression by liver DC, generally regarded as less immunostimulatory than secondary lymphoid tissue DC. Because the liver lies directly downstream from the gut, it is constantly exposed to bacterial LPS, a TLR4 ligand. We examined TLR4 expression by freshly isolated, flow-sorted C57BL/10 mouse liver DC compared with spleen DC. Real-time PCR revealed that liver CD11c+CD8α− (myeloid) and CD11c+CD8α+ (“lymphoid-related”) DC expressed lower TLR4 mRNA compared with their splenic counterparts. Lower TLR4 expression correlated with reduced capacity of LPS (10 ng/ml) but not anti-CD40-stimulated liver DC to induce naive allogeneic (C3H/HeJ) T cell proliferation. By contrast to LPS-stimulated splenic DC, these LPS-activated hepatic DC induced alloantigen-specific T cell hyporesponsiveness in vitro, correlated with deficient Th1 (IFN-γ) and Th2 (IL-4) responses. When higher LPS concentrations (≥100 ng/ml) were tested, the capacity of liver DC to induce proliferation of T cells and Th1-type responses was enhanced, but remained inferior to that of splenic DC. Hepatic DC activated by LPS in vivo were inferior allogeneic T cell stimulators compared with splenic DC, whereas adoptive transfer of LPS-stimulated (10 ng/ml) liver DC induced skewing toward Th2 responses. These data suggest that comparatively low expression of TLR4 by liver DC may limit their response to specific ligands, resulting in reduced or altered activation of hepatic adaptive immune responses.
TGF-β and Vitamin D3 Utilize Distinct Pathways to Suppress IL-12 Production and Modulate Rapid Differentiation of Human Monocytes into CD83+ Dendritic Cells
Authors: Lyakh Lyudmila A. , Sanford Michael , Chekol Sebel , Young Howard A. , Roberts Anita B.
Published: April 2014

We previously demonstrated that agents known to signal infection or inflammation can rapidly and directly drive differentiation of human CD14+ monocytes into CD83+ dendritic cells (DCs) when introduced to cells under serum-free conditions. In this study, we evaluated the effects of TGF-β and vitamin D3 (VitD3) on the proportion and function of monocytes that adopt DC characteristics. TGF-β significantly decreased the proportion of cells that rapidly adopted stable DC characteristics in response to LPS, but had little or no effect on calcium ionophore-induced differentiation. In contrast, VitD3 showed no such pathway specificity and dramatically suppressed differentiation of monocytes into DCs in response to these agents. Both TGF-β and VitD3 altered cytokine and chemokine production in LPS-treated monocytes, inhibited IL-12 and IL-10 secretion, and decreased the functional capacity of DCs. Despite the similar effects of TGF-β and VitD3, there are significant differences in the signaling pathways used by these agents, as evidenced by their distinct effects on LPS- and calcium ionophore-induced DC differentiation, on LPS-induced secretion of IL-10, and on two members of the NF-κB family of transcription factors, RelB and cRel. These studies identify TGF-β and VitD3 as potent regulatory factors that use distinct pathways to suppress both the differentiation of DCs as well as their capacity to secrete the Th1-polarizing cytokine IL-12. Because these agents are present in serum and negatively affect DC differentiation at physiological concentrations, our findings are likely to have significance regarding the in vivo role of TGF-β and VitD3 in determining the type of immune responses.
Distinct Effects of TGF-β1 on CD4+ and CD8+ T Cell Survival, Division, and IL-2 Production: A Role for T Cell Intrinsic Smad3
Authors: McKarns Susan C. , Schwartz Ronald H.
Published: April 2014

TGF-β1 is critical for maintaining T cell homeostasis. Smad3 has been implicated in this regulatory process, yet the cellular targets and molecular details remain poorly understood. In this study, we report that TGF-β1 impairs the entry of CD4+ and CD8+ T cells into the cell cycle as well as their progression through subsequent rounds of division, and show that Smad3 is essential for TGF-β1 to inhibit TCR-induced division of only CD4+ and not CD8+ T cells. Both CD8+ and CD4+ T cells from Smad3−/− mice were refractory to TGF-β1-induced inhibition of IL-2 production, thus demonstrating that not all CD8+ T cell responses to TGF-β1 are Smad3 independent. These TGF-β1 effects were all T cell intrinsic, as they were reproduced in purified CD4+ and CD8+ T cells. Finally, we found that Smad3 was critical for the survival of CD8+, but not CD4+ T cells following activation ex vivo. The TCR-induced death of Smad3−/− CD8+ T cells was not dependent upon TNF-α production. Exogenous TGF-β1 partially rescued the CD8+ T cells by signaling through a Smad3-independent pathway. TGF-β1 also enhanced survival of TCR-stimulated CD4+CD44high T cells in a Smad3-independent manner. Collectively, these findings firmly establish for the first time that TGF-β1 discriminately regulates CD4+ and CD8+ T cell expansion by signaling through distinct intracellular pathways.
The Role of MTJ-1 in Cell Surface Translocation of GRP78, a Receptor for α2-Macroglobulin-Dependent Signaling
Authors: Misra Uma Kant , Gonzalez-Gronow Mario , Gawdi Govind , Pizzo Salvatore Vincent
Published: April 2014

MTJ-1 associates with a glucose-regulated protein of Mr ∼78,000(GRP78) in the endoplasmic reticulum and modulates GRP78 activity as a chaperone. GRP78 also exists on the cell surface membrane, where it is associated with a number of functions. MHC class I Ags on the cell surface are complexed to GRP78. GRP78 also serves as the receptor for α2-macroglobulin-dependent signaling and for uptake of certain pathogenic viruses. The means by which GRP78, lacking a transmembrane domain, can fulfill such functions is unclear. In this study we have examined the question of whether MTJ-1, a transmembrane protein, is involved in the translocation of GRP78 to the cell surface. MTJ-1 and GRP78 coimmunoprecipitated from macrophage plasma membrane lysates. Silencing of MTJ-1 gene expression greatly reduced MTJ-1 mRNA and protein levels, but also abolished cell surface localization of GRP78. Consequently, binding of the activated and receptor-recognized form of α2-macroglobulin to macrophages was greatly reduced, and activated and receptor-recognized form of α2-macroglobulin-induced calcium signaling was abolished in these cells. In conclusion, we show that in addition to assisting the chaperone GRP78 in protein quality control in the endoplasmic reticulum, MTJ-1 is essential for transport of GRP78 to the cell surface, which serves a number of functions in immune regulation and signal transduction.
Membrane-Anchored β2-Microglobulin Stabilizes a Highly Receptive State of MHC Class I Molecules
Authors: Berko Dikla , Carmi Yaron , Cafri Gal , Ben-Zaken Shimrit , Sheikhet Helena Migalovich , Tzehoval Esther , Eisenbach Lea , Margalit Alon , Gross Gideon
Published: April 2014

The magnitude of response elicited by CTL-inducing vaccines correlates with the density of MHC class I (MHC-I)-peptide complexes formed on the APC membrane. The MHC-I L chain, β2-microglobulin (β2m), governs complex stability. We reasoned that genetically converting β2m into an integral membrane protein should exert a marked stabilizing effect on the resulting MHC-I molecules and enhance vaccine efficacy. In the present study, we show that expression of membranal human β2m (hβ2m) in mouse RMA-S cells elevates MHC-I thermal stability. RMA-S transfectants bind an exogenous peptide at concentrations 104- to 106-fold lower than parental RMA-S, as detected by complex-specific Abs and by T cell activation. Moreover, saturation of the transfectants’ MHC-I by exogenous peptide occurs within 1 min, as compared with ∼1 h required for parental cells. At saturation, however, level of peptide bound by modified cells is only 3- to 5-fold higher. Expression of native hβ2m only results in marginal effect on the binding profile. Soluble β2m has no effect on the accelerated kinetics, but the kinetics of transfectants parallel that of parental cells in the presence of Abs to hβ2m. Ab inhibition and coimmunoprecipitation analyses suggest that both prolonged persistence of peptide-receptive H chain/β2m heterodimers and fast heterodimer formation via lateral diffusion may contribute to stabilization. In vivo, peptide-loaded transfectants are considerably superior to parental cells in suppressing tumor growth. Our findings support the role of an allosteric mechanism in determining ternary MHC-I complex stability and propose membranal β2m as a novel scaffold for CTL induction.
Electrostatic Modeling Predicts the Activities of Orthopoxvirus Complement Control Proteins
Authors: Sfyroera Georgia , Katragadda Madan , Morikis Dimitrios , Isaacs Stuart N. , Lambris John D.
Published: April 2014

Regulation of complement activation by pathogens and the host are critical for survival. Using two highly related orthopoxvirus proteins, the vaccinia and variola (smallpox) virus complement control proteins, which differ by only 11 aa, but differ 1000-fold in their ability to regulate complement activation, we investigated the role of electrostatic potential in predicting functional activity. Electrostatic modeling of the two proteins predicted that altering the vaccinia virus protein to contain the amino acids present in the second short consensus repeat domain of the smallpox protein would result in a vaccinia virus protein with increased complement regulatory activity. Mutagenesis of the vaccinia virus protein confirmed that changing the electrostatic potential of specific regions of the molecule influences its activity and identifies critical residues that result in enhanced function as measured by binding to C3b, inhibition of the alternative pathway of complement activation, and cofactor activity. In addition, we also demonstrate that despite the enhanced activity of the variola virus protein, its cofactor activity in the factor I-mediated degradation of C3b does not result in the cleavage of the α′ chain of C3b between residues 954–955. Our data have important implications in our understanding of how regulators of complement activation interact with complement, the regulation of the innate immune system, and the rational design of potent complement inhibitors that might be used as therapeutic agents.
GATA-3 Is an Important Transcription Factor for Regulating Human NKG2A Gene Expression
Authors: Marusina Alina I. , Kim Dae-Ki , Lieto Louis D. , Borrego Francisco , Coligan John E.
Published: April 2014

CD94/NKG2A is an inhibitory receptor expressed by most human NK cells and a subset of T cells that recognizes HLA-E on potential target cells. To study the transcriptional regulation of the human NKG2A gene, we cloned a 3.9-kb genomic fragment that contains a 1.65-kb region upstream of the exon 1, as well as exon 1 (untranslated), intron 1 and exon 2. Using deletion mutants, we identified a region immediately upstream from the most upstream transcriptional initiation site that led to increased transcriptional activity from a luciferase reporter construct in YT-Indy (NKG2A positive) cells relative to Jurkat and K562 (both NKG2A negative) cells. We also localized a DNase I hypersensitivity site to this region. Within this 80-bp segment, we identified two GATA binding sites. Mutation of GATA binding site II (−2302 bp) but not GATA binding site I (−2332 bp) led to decreased transcriptional activity. Pull-down assays revealed that GATA-3 could bind oligonucleotide probes containing the wild type but not a mutated GATA site II. Using chromatin immunoprecipitation assays, we showed that GATA-3 specifically binds to the NKG2A promoter in situ in NKL and primary NK cells, but not in Jurkat T cells. Moreover, coexpression of human GATA-3 with an NKG2A promoter construct in K562 cells led to enhanced promoter activity, and transfection of NKL cells with small interfering RNA specific for GATA-3 reduced NKG2A cell surface expression. Taken together, our data indicate that GATA-3 is an important transcription factor for regulating NKG2A gene expression.
Hepatitis C Virus Core Protein Modulates TRAIL-Mediated Apoptosis by Enhancing Bid Cleavage and Activation of Mitochondria Apoptosis Signaling Pathway
Authors: Chou Ai-Hsiang , Tsai Hwei-Fang , Wu Yi-Ying , Hu Chung-Yi , Hwang Lih-Hwa , Hsu Ping-I. , Hsu Ping-Ning
Published: April 2014

Hepatitis C virus (HCV) is a major human pathogen causing chronic liver disease, which leads to cirrhosis of liver and hepatocellular carcinoma. The HCV core protein, a viral nucleocapsid, has been shown to affect various intracellular events, including cell proliferation and apoptosis. However, the precise mechanisms of the effects are not fully understood. In this study, we show that HCV core protein sensitizes human hepatocellular carcinoma cell line, Huh7, conferred sensitivity to TRAIL-, but not Fas ligand-mediated apoptosis. Huh7 cells are resistant to TRAIL, despite the induction of caspase-8 after TRAIL engagement. However, HCV core protein induces TRAIL apoptosis signaling via sequential induction of caspase-8, Bid cleavage, activation of mitochondrial pathway, and effector caspase-3. HCV core protein also induces activation of caspase-9 after TRAIL engagement, and the induction of TRAIL sensitivity by HCV core protein could be reversed by caspase-9 inhibitor. Therefore, the HCV core protein-induced TRAIL-mediated apoptosis is dependent upon activation of caspase-8 downstream pathway to convey the death signal to mitochondria, leading to activation of mitochondrial signaling pathway and breaking the apoptosis resistance. These results combined indicate that the HCV core protein enhances TRAIL-, but not Fas ligand-mediated apoptotic cell death in Huh7 cells via a mechanism dependent on the activation of mitochondria apoptosis signaling pathway. These results suggest that HCV core protein may have a role in immune-mediated liver cell injury by modulation of TRAIL-induced apoptosis.
Vaccine-Elicited Antibodies Mediate Antibody-Dependent Cellular Cytotoxicity Correlated with Significantly Reduced Acute Viremia in Rhesus Macaques Challenged with SIVmac251
Authors: Gómez-Román V. Raúl , Patterson L. Jean , Venzon David , Liewehr David , Aldrich Kris , Florese Ruth , Robert-Guroff Marjorie
Published: April 2014

Effector cells armed with Abs can eliminate virus-infected target cells by Ab-dependent cellular cytotoxicity (ADCC), an immune mechanism that has been largely overlooked in HIV vaccine development. Here, we show that a prime/boost AIDS vaccine approach elicits potent ADCC activity correlating with protection against SIV in rhesus macaques (Macacca mulatta). Priming with replicating adenovirus type 5 host range mutant-SIV recombinants, followed by boosting with SIV gp120, elicited Abs with ADCC activity against SIVmac251-infected cells. In vitro ADCC activity correlated with in vivo reduced acute viremia after a mucosal challenge with pathogenic SIV. Our findings expose ADCC activity as an immune correlate that may be relevant in the rational design of an efficacious vaccine against HIV.
A Novel Neurotoxoid Vaccine Prevents Mucosal Botulism
Authors: Kobayashi Ryoki , Kohda Tomoko , Kataoka Kosuke , Ihara Hideshi , Kozaki Shunji , Pascual David W. , Staats Herman F. , Kiyono Hiroshi , McGhee Jerry R. , Fujihashi Kohtaro
Published: April 2014

The threat posed by botulism, classically a food- and waterborne disease with a high morbidity and mortality, has increased exponentially in an age of bioterrorism. Because botulinum neurotoxin (BoNT) could be easily disseminated by terrorists using an aerosol or could be used to contaminate the food or water supply, the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases has classified it as a category A agent. Although clearly the development of a safe and effective mucosal vaccine against this toxin should be a high priority, essentially no studies to date have assessed mucosal immune responses to this disease. To bridge this gap in our knowledge, we immunized mice weekly for 4 wk with nasal doses of BoNT type A toxoid and a mutant of cholera toxin termed E112K. We found elevated levels of BoNT-specific IgG Abs in plasma and of secretory IgA Abs in external secretions (nasal washes, saliva, and fecal extracts). When mice given nasal BoNT vaccine were challenged with 4 × 103 LD50 of BoNT type A (BoNT/A) via the i.p. route, complete protection was seen, while naive mice given the same dosage died within 2 h. To further confirm the efficacy of this nasal BoNT vaccine, an oral LD50 was determined. When mice were given an oral challenge of 5 μg (2 × oral LD50) of progenitor BoNT/A, all immunized mice survived beyond 5 days, while nonimmunized mice did not. The fecal extract samples from nasally vaccinated mice were found to contain neutralizing secretory IgA Abs. Taken together, these results show that nasal BoNT/A vaccine effectively prevents mucosal BoNT intoxication.
Identification of Murine Poxvirus-Specific CD8+ CTL Epitopes with Distinct Functional Profiles
Authors: Mathew Anuja , Terajima Masanori , West Kim , Green Sharone , Rothman Alan L. , Ennis Francis A. , Kennedy Jeffrey S.
Published: April 2014

Murine T cell epitopes against vaccinia virus (VV) have not been characterized to date in part due to the large and complex genome of VV. We have identified and characterized two CD8+ T cell epitopes on the A47L (modified VV Ankara strain (MVA)-029) and J6R (MVA-043) proteins of VV that are Db and Kb restricted, respectively. Following i.p. immunization with VV New York City Board of Health (NYCBH) strain, MVA-029 peptide-stimulated splenocytes secreted IFN-γ from 7 days to 7 mo postimmunization, and virus-stimulated effectors were also able to lyse MVA-029-pulsed target cells at the same time points. In contrast, MVA-043 peptide-stimulated splenocytes secreted very low levels of IFN-γ only at day 7 but maintained the ability to lyse target cells up to 2 mo postimmunization. Both MVA-029 and MVA-043 peptide-stimulated lymph node cells degranulated similarly as assessed by Ag-induced CD107 expression. T cell responses to whole-virus stimulation remained robust and steady during the acute and memory T cell response to VV. Identification of T cell epitopes on VV will enable further studies to increase our understanding of the role of CD8+ T cells in VV infection and assist in the design of new protective strategies.