bluuebunny/crossref_metadata_2025 huggingface dataset:
CpG Oligodeoxynucleotides Protect Newborn Mice from a Lethal Challenge with the Neurotropic Tacaribe Arenavirus
Authors: Pedras-Vasconcelos João A. , Goucher David , Puig Montserrat , Tonelli Leonardo H. , Wang Vivian , Ito Shuichi , Verthelyi Daniela
Published: April 2014

The innate immune system is key to limiting the early spread of most pathogens and directing the development of Ag-specific immunity. Recently, a number of synthetic molecules that activate the innate immune system by stimulating TLRs have been identified. Among them, synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs (CpG ODNs) were shown to activate TLR9-bearing B cells, macrophages, and dendritic cells to induce a strong proinflammatory milieu and a type 1-biased immune response that protects mice from a variety of parasitic, bacterial, and viral infections. Although the protective effect of CpG ODN in adult mice was well established, its effectiveness in neonates, which have lower numbers of dendritic, B, and T cells and tend to favor Th2 responses, was unclear. This study uses the New World arenavirus Tacaribe, a neurotropic pathogen that is lethal in newborn mice, to explore the effectiveness of TLR-mediated innate immune responses. Neonatal BALB/c mice treated with CpG ODN at the time of infection had reduced viral load (p < 0.01) and increased survival (52%, p < 0.001 i.p.; 36%, p < 0.05 intranasally). Protection was achieved in mice treated no later than 3 days postchallenge and appears to be mediated by an increase in Ag-specific Abs (IgG and IgM) and to require inducible NO synthase expression and NO production. To our knowledge, this is the first study assessing the mechanisms by which CpG ODN can protect mice from a neurotropic viral infection.
Duration and Intensity of NF-κB Activity Determine the Severity of Endotoxin-Induced Acute Lung Injury
Authors: Everhart M. Brett , Han Wei , Sherrill Taylor P. , Arutiunov Melissa , Polosukhin Vasiliy V. , Burke James R. , Sadikot Ruxana T. , Christman John W. , Yull Fiona E. , Blackwell Timothy S.
Published: April 2014

Activation of innate immunity in the lungs can lead to a self-limited inflammatory response or progress to severe lung injury. We investigated whether specific parameters of NF-κB pathway activation determine the outcome of acute lung inflammation using a novel line of transgenic reporter mice. Following a single i.p. injection of Escherichia coli LPS, transient NF-κB activation was identified in a variety of lung cell types, and neutrophilic inflammation resolved without substantial tissue injury. However, administration of LPS over 24 h by osmotic pump (LPS pump) implanted into the peritoneum resulted in sustained, widespread NF-κB activation and neutrophilic inflammation that culminated in lung injury at 48 h. To determine whether intervention in the NF-κB pathway could prevent progression to lung injury in the LPS pump model, we administered a specific IκB kinase inhibitor (BMS-345541) to down-regulate NF-κB activation following the onset of inflammation. Treatment with BMS-345541 beginning at 20 h after osmotic pump implantation reduced lung NF-κB activation, concentration of KC and MIP-2 in lung lavage, neutrophil influx, and lung edema measured at 48 h. Therefore, sustained NF-κB activation correlates with severity of lung injury, and interdiction in the NF-κB pathway is beneficial even after the onset of lung inflammation.
In Vitro Activation of CD8 Interphotoreceptor Retinoid-Binding Protein-Specific T Cells Requires not only Antigenic Stimulation but also Exogenous Growth Factors
Authors: Peng Yong , Shao Hui , Ke Yan , Zhang Ping , Xiang Jim , Kaplan Henry J. , Sun Deming
Published: April 2014

In a previous study, we demonstrated that immunization with the uveitogenic peptide interphotoreceptor retinoid-binding protein (IRBP) 1–20 induces both CD4 and CD8 uveitogenic T cells in the B6 mouse. In the current study, we determined the role of the CD8 IRBP-specific T cells in the pathogenesis of experimental autoimmune uveitis. We also determined the conditions that facilitated the activation of CD8 autoreactive T cells. Our results showed that the β2-microglobulin−/− mouse had a greatly decreased susceptibility to induction of experimental autoimmune uveitis by adoptive transfer of IRBP-specific T cells from B6 mice. We also showed that unlike CD4 autoreactive T cells, activated CD8 autoreactive T cells produced only a limited number and amounts of growth factors. As a result, in the absence of exogenously supplied growth factor(s), CD8 T cell activation and expansion were aborted. However, the growth and expansion of triggered CD8 autoreactive T cells could be supported by various cytokines. In addition to factors produced by activated CD4 autoreactive T cells, factors produced by nonlymphoid cells, such as IL-7 and IL-15, and unidentified factors in the culture supernatants of astrocytes and retinal pigment epithelial cells support the CD8 autoreactive T cells as well. Finally, we showed that, although several cytokines augmented the CD8 T cell response in vitro, different cytokines appeared to act on different CD8 subsets or on different activation/differentiation phases of CD8 autoreactive T cells. As a result, cytokines, such as IL-7, supported the proliferation and survival of CD8 IRBP-specific T cells, while others had only a growth-promoting effect.
Histone Hyperacetylation Is Associated with Amelioration of Experimental Colitis in Mice
Authors: Glauben Rainer , Batra Arvind , Fedke Inka , Zeitz Martin , Lehr Hans A. , Leoni Flavio , Mascagni Paolo , Fantuzzi Giamila , Dinarello Charles A. , Siegmund Britta
Published: April 2014

Inhibitors of histone deacetylases (HDAC) are being studied for their antiproliferative effects in preclinical cancer trials. Recent studies suggest an anti-inflammatory role for this class of compounds. Because inflammatory bowel disease is associated with an increased risk of malignancies, agents with antiproliferative and anti-inflammatory properties would be of therapeutic interest. HDAC inhibitors from various classes were selected and evaluated for their in vitro capacity to suppress cytokine production and to induce apoptosis and histone acetylation. Valproic acid (VPA) and suberyolanilide hydroxamic acid (SAHA) were chosen for further studies in dextran sulfate sodium- and trinitrobenzene sulfonic acid-induced colitis in mice. In vitro, inhibition of HDAC resulted in a dose-dependent suppression of cytokine synthesis and apoptosis induction requiring higher concentrations of HDAC inhibitors for apoptosis induction compared with cytokine inhibition. Oral administration of either VPA or SAHA reduced disease severity in dextran sulfate sodium-induced colitis. The macroscopic and histologic reduction of disease severity was associated with a marked suppression of colonic proinflammatory cytokines. In parallel to the beneficial effect observed, a dose-dependent increase in histone 3 acetylation at the site of inflammation was shown under VPA treatment. Furthermore, SAHA as well as VPA treatment resulted in amelioration of trinitrobenzene sulfonic acid-induced colitis, which was associated with an increase of apoptosis of lamina propria lymphocytes. Inhibitors of HDAC reveal strong protective effects in different models of experimental colitis by inducing apoptosis and suppressing proinflammatory cytokines, thereby representing a promising class of compounds for clinical studies in human inflammatory bowel disease.
Ovarian Cancer Cells Polarize Macrophages Toward A Tumor-Associated Phenotype
Authors: Hagemann Thorsten , Wilson Julia , Burke Frances , Kulbe Hagen , Li Ninfeng Fiona , Plüddemann Annette , Charles Kellie , Gordon Siamon , Balkwill Frances R.
Published: April 2014

Tumor-associated macrophages (TAM) may have tumor-promoting activity, but it is not clear how their phenotype is achieved. In this study, we demonstrate that ovarian cancer cells switch cocultured macrophages to a phenotype similar to that found in ovarian tumors. Tumor cells caused dynamic changes in macrophage cytokine, chemokine, and matrix metalloprotease mRNA, and protein-inducing mediators that are found in human cancer. Macrophage mannose, mannose receptor, and scavenger receptors (SR-As) were also up-regulated by coculture, but not by conditioned medium. To further validate the model, we studied SR-A regulation on TAM in vitro and in vivo. Coculture of murine macrophages from mice deficient in TNF-α or its receptors revealed that TNF-α was key to SR-A induction via its p75 receptor. SR-A expression was also reduced in TAM from ovarian cancers treated with anti-TNF-α Abs or grown in TNF-α−/− mice. Chemical communication between tumor cells and macrophages may be important in regulating the cancer cytokine microenvironment.
A Severe Form of Human Combined Immunodeficiency Due to Mutations in DNA Ligase IV
Authors: Enders Anselm , Fisch Paul , Schwarz Klaus , Duffner Ulrich , Pannicke Ulrich , Nikolopoulos Elisabeth , Peters Anke , Orlowska-Volk Marzenna , Schindler Detlev , Friedrich Wilhelm , Selle Barbara , Niemeyer Charlotte , Ehl Stephan
Published: April 2014

DNA ligase IV (LigIV) deficiency was identified as the molecular basis for a severe form of combined immunodeficiency in two microcephalic siblings with cellular radiosensitivity. In one patient the diagnosis was made directly after birth, allowing analysis of the role of LigIV in the development of specific immune cells. Absolute numbers of B cells were reduced 100-fold and αβ T cells 10-fold, whereas γδ T cells were normal. Spectratyping of all three cell populations showed a diverse repertoire, but sequencing of IgH V(D)J junctions revealed shorter CDR3 regions due to more extensive nucleotide deletions among D and J elements and fewer N nucleotide insertions. Clonal restriction of IgG-expressing, but not IgM-expressing, B cells and the lack of primary and secondary lymph node follicles indicated impaired class switch recombination. Observations in the older sibling showed that this rudimentary immune system was able to mount specific responses to infection. However, partial Ab responses and extensive amplification of γδ T cells could not prevent a life-threatening course of viral and bacterial infections, the development of an EBV-induced lymphoma, and immune dysregulation reflected by severe autoimmune cytopenia. Impaired generation of immune diversity under conditions of limited LigIV activity can cause a human SCID variant with a characteristic immunological phenotype.
Allergen-Specific MHC Class II Tetramer+ Cells Are Detectable in Allergic, but Not in Nonallergic, Individuals
Authors: Macaubas Claudia , Wahlstrom Jan , Galvão da Silva Ana Paula , Forsthuber Thomas G. , Sønderstrup Grete , Kwok William W. , DeKruyff Rosemarie H. , Umetsu Dale T.
Published: April 2014

Allergen-specific cells are present in very low frequency in peripheral blood of humans, and differ in function in allergic and nonallergic individuals. We report in this study that soluble class II MHC tetramers can be used to directly identify and study such allergen epitope-specific CD4+ T cells in humans. We identified the major antigenic epitope of rye grass allergen Lol p 1 in HLA-DRB1*0401 individuals using HLA-DR*0401 transgenic mice and peripheral blood cells from HLA-DR*0401 individuals. Using DRB1*0401 tetramers loaded with this major epitope of Lol p 1, we detected allergen-specific CD4+ T cells in the peripheral blood of DRB1*0401 rye grass allergic individuals after ex vivo expansion with allergen. These tetramer-positive cells produced IL-4, but little IFN-γ. In contrast, we were unable to detect rye grass tetramer-positive cells in cultures from HLA-DR*0401 nonallergic individuals, even after expansion with IL-2. Thus, our results suggest that rye grass allergen-specific T cells in DR*0401 nonallergic subjects are present at very low levels (e.g., because of deletion or suppression), differ in a fundamental way in their requirement for ex vivo expansion (e.g., they may be anergic), or use TCRs distinct from those of allergic individuals. Thus, analysis using DRB1*0401 tetramers loaded with a major epitope of Lol p 1 indicates that allergen-specific CD4+ T cells in nonallergic individuals are distinct from those in allergic subjects.
Identification of Novel Subdominant Epitopes on the Carcinoembryonic Antigen Recognized by CD4+ T Cells of Lung Cancer Patients
Authors: Crosti Mariacristina , Longhi Renato , Consogno Giuseppe , Melloni Giulio , Zannini Piero , Protti Maria Pia
Published: April 2014

The carcinoembryonic Ag (CEA) is an attractive target for immunotherapy because of its expression profile and role in tumor progression. To verify the existence of spontaneous anti-CEA CD4+ T cells in lung cancer patients, we first identified CEA sequences forming naturally processed epitopes, and then used the identified epitopes to test their recognition by CD4+ T cells from the patients. We had previously identified CEA177–189/355–367 as an immunodominant epitope recognized by CD4+ T cells in association with several HLA-DR alleles. In this study, we identified four additional subdominant CEA sequences (CEA99–111, CEA425–437, CEA568–582, and CEA666–678), recognized in association with one or more HLA-DR alleles. Peptide-specific CD4+ T cells produced proinflammatory cytokines when challenged with the native protein and CEA-expressing tumor cells, thus demonstrating that the identified CEA sequences contain naturally processed epitopes. However, CEA is expressed in the thymus and belongs to the CD66 family that comprises highly homologous molecules expressed on hemopoietic cells, raising concerns about tolerance interfering with the in vivo development of anti-CEA immunity. We thus tested the spontaneous reactivity to the identified epitopes of peripheral blood CD4+ T lymphocytes from eight early-stage lung cancer patients bearing CEA-positive tumors. We found GM-CSF- and IFN-γ- producing CD4+ T cells in two patients. Our data indicate that CD4+ immune responses against CEA develop in neoplastic patients, suggesting that tolerance toward CEA or cross-reactive CD66 homologous molecules might be either not absolute or be overcome in the neoplastic disease.
BCR/ABL Oncogene Directly Controls MHC Class I Chain-Related Molecule A Expression in Chronic Myelogenous Leukemia
Authors: Boissel Nicolas , Rea Delphine , Tieng Vannary , Dulphy Nicolas , Brun Manuel , Cayuela Jean-Michel , Rousselot Philippe , Tamouza Ryad , Le Bouteiller Philippe , Mahon François-Xavier , Steinle Alexander , Charron Dominique , Dombret Hervé , Toubert Antoine
Published: April 2014

MHC class I chain-related molecules (MIC) participate in immune surveillance of cancer through engagement of the NKG2D-activating receptor on NK and T cells. Decreased NKG2D expression and function upon chronic exposure to NKG2D ligands and/or soluble forms of MIC (sMIC) may participate in immune escape. In chronic myeloid leukemia, a malignancy caused by the BCR/ABL fusion oncoprotein, we showed cell surface expression of MICA on leukemic, but not healthy, donor hemopoietic CD34+ cells. At diagnosis, chronic myeloid leukemia patients had abnormally high serum levels of sMICA and weak NKG2D expression on NK and CD8+ T cells, which were restored by imatinib mesylate (IM) therapy. In the BCR/ABL+ cell line K562, IM decreased both surface MICA/B expression and NKG2D-mediated lysis by NK cells. Silencing BCR/ABL gene expression directly evidenced its role in the control of MICA expression. IM did not affect MICA mRNA levels, but decreased MICA protein production and release. Sucrose density gradient fractionation of K562 cytoplasmic extracts treated with IM showed a shift in the distribution of MICA mRNA from the polysomal toward the monosomal fractions, consistent with decreased translation. Among the major pathways activated by BCR/ABL that regulate translation, PI3K and mammalian target of rapamycin were shown to control MICA expression. These data provide evidence for direct control of MICA expression by an oncogene in human malignancy and indicate that posttranscriptional mechanisms may participate in the regulation of MICA expression.
Cutting Edge: Cognate CD4 Help Promotes Recruitment of Antigen-Specific CD8 T Cells around Dendritic Cells
Authors: Beuneu Hélène , Garcia Zacarias , Bousso Philippe
Published: April 2014

The cellular orchestration underlying help provided by CD4 T lymphocytes to CD8 T cell responses is not fully understood. We documented that the formation of three-cell clusters occurred as soon as day 1 and relied on long-lasting CD4 and CD8 T cell interactions with dendritic cells (DCs). The influence of CD4 help on CD8 T cell differentiation could be observed as early as the second round of cell division. Importantly, our results identify a new facet to the phenomenon of CD4 help in which DCs, upon cognate interactions with CD4 T cells, increase their ability to attract/retain Ag-specific CD8 T cells. Our results support a model in which CD4 help operates rapidly, in part by favoring CD8 T cells recruitment around those DCs that are the most competent for priming.
Cutting Edge: Central Memory T Cells Do Not Show Accelerated Proliferation or Tissue Infiltration in Response to Localized Herpes Simplex Virus-1 Infection
Authors: Stock Angus T. , Jones Claerwen M. , Heath William R. , Carbone Francis R.
Published: April 2014

Memory T cells mount an enhanced response to secondary infections. Such an enhancement has been attributed in part to the ability of memory cells to more rapidly respond to cognate stimulation. In this study we have examined the rapidity with which murine CD8+ memory T cells respond to a localized infection with HSV. Although central memory T cells (TcM), but not the effector memory T cells, mounted a strong recall response to secondary infection, the kinetics of TcM proliferation, the magnitude of their expansion, and their infiltration into infected nonlymphoid tissues were not advanced compared with that observed for naive T cells. These findings imply that it is the lack of accelerated proliferation kinetics and the subsequent delayed dissemination into the periphery that limits the ability of TcM to rapidly control localized virus replication.
Cutting Edge: IFN-γ Regulates the Induction and Expansion of IL-17-Producing CD4 T Cells during Mycobacterial Infection
Authors: Cruz Andrea , Khader Shabaana A. , Torrado Egidio , Fraga Alexandra , Pearl John E. , Pedrosa Jorge , Cooper Andrea M. , Castro António G.
Published: April 2014

T cell responses are important to the control of infection but are deleterious if not regulated. IFN-γ-deficient mice infected with mycobacteria exhibit enhanced accumulation of activated effector T cells and neutrophils within granulomatous lesions. These cells do not control bacterial growth and compromise the integrity of the infected tissue. We show that IFN-γ-deficient mice have increased numbers of IL-17-producing T cells following infection with Mycobacterium bovis bacille Calmette Guérin. Furthermore, exogenous IFN-γ increases IL-12 and decreases IL-23 production by bacille Calmette Guérin-infected bone marrow-derived dendritic cells and reduces the frequency of IL-17-producing T cells induced by these bone marrow-derived dendritic cells. These data support the hypothesis that, during mycobacterial infection, both IFN-γ- and IL-17-producing T cells are induced, but that IFN-γ serves to limit the IL-17-producing T cell population. This counterregulation pathway may be an important factor in limiting mycobacterially associated immune-mediated pathology.
EBV-Specific CD4+ T Cell Clones Exhibit Vigorous Allogeneic Responses
Authors: Landais Elise , Morice Alexis , Long Heather M. , Haigh Tracey A. , Charreau Béatrice , Bonneville Marc , Taylor Graham S. , Houssaint Elisabeth
Published: April 2014

Alloreactive T cells play a key role in mediating graft-vs-host disease and allograft rejection, and recent data suggest that most T cell alloreactivity resides within the CD4 T cell subset. Particularly, T cell responses to herpesvirus can shape the alloreactive repertoire and influence transplantation outcomes. In this study, we describe six distinct EBV-specific CD4+ T cell clones that cross-reacted with EBV-transformed lymphoblastoid cell lines (LCLs), dendritic cells, and endothelial cells expressing MHC class II alleles commonly found in the population. Allorecognition showed exquisite MHC specificity. These CD4+ T cell clones efficiently killed dendritic cells or LCLs expressing the cross-reactive allogeneic MHC class II molecules, whereas they did not kill autologous LCLs. Endothelial cells expressing the proper allogeneic MHC molecules were poorly killed, but they induced high-level TNF-α production by the EBV-specific CD4+ T cell clones. As already proposed, the strong alloreactivity toward LCLs suggest that these cells could be used for selective depletion of alloreactive T cells.
Tripeptidyl Peptidase II Is the Major Peptidase Needed to Trim Long Antigenic Precursors, but Is Not Required for Most MHC Class I Antigen Presentation
Authors: York Ian A. , Bhutani Nidhi , Zendzian Sophia , Goldberg Alfred L. , Rock Kenneth L.
Published: April 2014

Recent reports concluded that tripeptidyl peptidase (TPPII) is essential for MHC class I Ag presentation and that the proteasome in vivo mainly releases peptides 16 residues or longer that require processing by TPPII. However, we find that eliminating TPPII from human cells using small interfering RNA did not decrease the overall supply of peptides to MHC class I molecules and reduced only modestly the presentation of SIINFEKL from OVA, while treatment with proteasome inhibitors reduced these processes dramatically. Purified TPPII digests peptides from 6 to 30 residues long at similar rates, but eliminating TPPII in cells reduced the processing of long antigenic precursors (14–17 residues) more than short ones (9–12 residues). Therefore, TPPII appears to be the major peptidase capable of processing proteasome products longer than 14 residues. However, proteasomes in vivo (like purified proteasomes) release relatively few such peptides, and these peptides processed by TPPII require further trimming in the endoplasmic reticulum (ER) by ER aminopeptidase 1 for presentation. Taken together, these observations demonstrate that TPPII plays a specialized role in Ag processing and one that is not essential for the generation of most presented peptides. Moreover, these findings reveal that three sequential proteolytic steps (by proteasomes, TPPII, and then ER aminopepsidase 1) are required for the generation of a subset of epitopes.
Reconstitution of Allogeneic Hemopoietic Stem Cells: The Essential Role of FcRγ and the TCR β-Chain-FCp33 Complex
Authors: Taylor Kendra N. , Shinde Patil Vivek R. , Colson Yolonda L.
Published: April 2014

Transplantation of purified allogeneic hemopoietic stem cells (SC) alone is characterized by a decreased risk of graft-vs-host disease but increased incidence of engraftment failure. It has been established that the facilitating cell (FC) promotes allogeneic SC reconstitution and results in donor-specific transplantation tolerance across MHC disparities, without graft-vs-host disease. Although the requirements for this facilitating function are not well-characterized, it is known that facilitation is dependent on FC expression of a unique heterodimer consisting of the TCR β-chain (TCRβ) and a 33-kDa protein, FCp33. The current study confirms that CD3ε and TCRβ expression are present on the FC at the time of transplantation and demonstrates that the majority of cells in the FC population express the TCR signaling molecule, FcRγ, rather than the more conventional CD3ζ receptor. Of particular significance, we have now demonstrated that FC-mediated allogeneic SC reconstitution is critically dependent on FcRγ expression and that FcRγ coprecipitates with the TCRβ-FCp33 heterodimer. The mandatory requirement of TCRβ and FcRγ for FC function provides the first evidence of a previously undescribed role for FcRγ in the facilitation of allogeneic SC reconstitution and establishes that FcRγ is part of the TCRβ-FCp33 complex uniquely expressed on FC.
Dendritic Cells Can Turn CD4+ T Lymphocytes into Vascular Endothelial Growth Factor-Carrying Cells by Intercellular Neuropilin-1 Transfer
Authors: Bourbié-Vaudaine Sarah , Blanchard Nicolas , Hivroz Claire , Roméo Paul-Henri
Published: April 2014

Neuropilin-1 (NRP1) is a transmembrane protein expressed on neuronal and endothelial cells where it plays a crucial role in guiding axons and regulating angiogenesis. We have recently shown that NRP1 also is expressed on dendritic cells (DC) in the human immune system and have proposed a role for NRP1 in the first stages of the immune response. In these studies, we show that NRP1 can be transferred with a high efficiency from human DC to T lymphocytes by trogocytosis. The NRP1 transfer can occur independently of T lymphocyte activation; the amount of NRP1 transferred depends on the NRP1 expression level on APC and is enhanced when T cells are activated through the TCR. Moreover, the NRP1 transfer occurs between specific donor and recipient cells, because no NRP1 transfer is observed between endothelial cells and T lymphocytes or between APCs and CD34+ hemopoietic cells. Finally, we show that a major NRP1 ligand, vascular endothelial growth factor (VEGF)165, is secreted by mature human DCs and binds to NRP1 captured by T lymphocytes. These results show that NRP1 transfer to T lymphocytes during the immune synapse can convert T lymphocytes into VEGF165-carrying cells. Together with the enhanced signaling of VEGF-R2 on endothelial cells in the presence, in trans, of the NRP1–VEGF165 complex, our results suggest that the intercellular transfer of NRP1 might participate in the Ag-independent remodelling of the endothelial vessels in secondary lymphoid organs during inflammation.
The Cytoplasmic Domain of Fas Ligand Costimulates TCR Signals
Authors: Sun Mingyi , Ames Kristina T. , Suzuki Ivy , Fink Pamela J.
Published: April 2014

Productive T cell activation generally requires costimulation in addition to a signal delivered through the TCR. Although FasL is well-characterized for its capacity to deliver a death signal through Fas, this TNF family member can also transmit a reverse signal to enhance Ag-driven T cell proliferation. In this study, we define this reverse signal through FasL as costimulation by showing it requires TCR coengagement and is CD28 independent. We demonstrate that FasL-mediated costimulation drives FasL recruitment into lipid rafts and association with select Src homology 3 (SH3)-containing proteins. We further show that the proline-rich intracellular domain of FasL is sufficient to costimulate by enhancing the phosphorylation of Akt, ERK1/2, JNK, and FasL itself, by activating the transcription factors NFAT and AP-1, and by enhancing IFN-γ production. These results elucidate the pathway of costimulation through the death inducer FasL, and comprise the first mechanistic analysis of a newly emerging group of costimulators, the TNF family.
Delayed Expansion and Contraction of CD8+ T Cell Response during Infection with VirulentSalmonella typhimurium
Authors: Luu Rachel A. , Gurnani Komal , Dudani Renu , Kammara Rajagopal , van Faassen Henk , Sirard Jean-Claude , Krishnan Lakshmi , Sad Subash
Published: April 2014

Ag presentation to CD8+ T cells often commences immediately after infection, which facilitates their rapid expansion and control of infection. Subsequently, the primed cells undergo rapid contraction. We report that this paradigm is not followed during infection with virulent Salmonella enterica, serovar Typhimurium (ST), an intracellular bacterium that replicates within phagosomes of infected cells. Although susceptible mice die rapidly (∼7 days), resistant mice (129×1SvJ) harbor a chronic infection lasting ∼60–90 days. Using rOVA-expressing ST (ST-OVA), we show that T cell priming is considerably delayed in the resistant mice. CD8+ T cells that are induced during ST-OVA infection undergo delayed expansion, which peaks around day 21, and is followed by protracted contraction. Initially, ST-OVA induces a small population of cycling central phenotype (CD62LhighIL-7RαhighCD44high) CD8+ T cells. However, by day 14–21, majority of the primed CD8+ T cells display an effector phenotype (CD62LlowIL-7RαlowCD44high). Subsequently, a progressive increase in the numbers of effector memory phenotype cells (CD62LlowIL-7RαhighCD44high) occurs. This differentiation program remained unchanged after accelerated removal of the pathogen with antibiotics, as majority of the primed cells displayed an effector memory phenotype even at 6 mo postinfection. Despite the chronic infection, CD8+ T cells induced by ST-OVA were functional as they exhibited killing ability and cytokine production. Importantly, even memory CD8+ T cells failed to undergo rapid expansion in response to ST-OVA infection, suggesting a delay in T cell priming during infection with virulent ST-OVA. Thus, phagosomal lifestyle may allow escape from host CD8+ T cell recognition, conferring a survival advantage to the pathogen.
Immunoediting of Cancers May Lead to Epithelial to Mesenchymal Transition
Authors: Knutson Keith L. , Lu Hailing , Stone Brad , Reiman Jennifer M. , Behrens Marshall D. , Prosperi Christine M. , Gad Ekram A. , Smorlesi Arianna , Disis Mary L.
Published: April 2014

Tumors evade both natural and pharmacologically induced (e.g., vaccines) immunity by a variety of mechanisms, including induction of tolerance and immunoediting. Immunoediting results in reshaping the immunogenicity of the tumor, which can be accompanied by loss of Ag expression and MHC molecules. In this study, we evaluated immunoediting in the neu-transgenic mouse model of breast cancer. A tumor cell line that retained expression of rat neu was generated from a spontaneous tumor of the neu-transgenic mouse and, when injected into the non-transgenic parental FVB/N mouse, resulted in the development of a strong immune response, initial rejection, and ultimately the emergence of neu Ag-loss variants. Morphologic and microarray data revealed that the immunoedited tumor cells underwent epithelial to mesenchymal transition accompanied by an up-regulation of invasion factors and increased invasiveness characteristic of mesenchymal tumor cells. These results suggest that immunoediting of tumor results in cellular reprogramming may be accompanied by alterations in tumor characteristics including increased invasive potential. Understanding the mechanisms by which tumors are immunoedited will likely lead to a better understanding of how tumors evade immune detection.
Differential Polarization of Immune Responses by Plant 2S Seed Albumins, Ber e 1, and SFA8
Authors: Kean Dorothy E. , Goodridge Helen S. , McGuinness Stephen , Harnett Margaret M. , Alcocer Marcos J. C. , Harnett William
Published: April 2014

The plant 2S seed albumins Ber e 1 and SFA8, although structurally very similar, vary with respect to their allergenic properties. Whereas the former represents a major allergen, the latter appears to promote only weak allergenic responses. The aim of this investigation was to determine whether the allergenic properties of Ber e 1 and SFA8 reflected differential polarization of dendritic cell (DC) and Th cell responses. We thus investigated the effect of recombinant forms of both allergens on DC and Th cell responses as indicated by cell surface phenotype and cytokine production. Exposure of murine DCs to SFA8, but not Ber e 1, resulted in production of the cytokines IL-12 p40 and TNF-α by a mechanism independent of recognition by TLRs. Furthermore, depending on the mouse strain used, increased expression of MHC class II and costimulatory molecules such as CD40, CD80, and CD86 was associated with exposure to SFA8, but not Ber e 1. In coculture experiments using the DO11.10 transgenic T cell that recognizes OVA peptide, DCs exposed to both allergens induced T cells to produce IFN-γ, but only Ber e 1 could induce significant production of IL-4 and IL-5. Likewise, analysis of transcription factors shows increased T-bet with respect to both allergens, but also GATA-3 with respect to Ber e 1. Overall, our data are consistent with the idea that the ability of Ber e 1, but not SFA8, to act as a potent allergen may reflect differences in their ability to induce IL-12 production.
Kinetics of B Cell Receptor Signaling in Human B Cell Subsets Mapped by Phosphospecific Flow Cytometry
Authors: Irish Jonathan M. , Czerwinski Debra K. , Nolan Garry P. , Levy Ronald
Published: April 2014

Differences in BCR signaling may govern outcomes as diverse as proliferation and cell death. We profiled BCR signaling kinetics in subsets of primary human B cells using flow cytometry. In the predominant population expressing IgM, BCR cross-linking led to a quick burst of Syk, ERK1/2, and p38 signaling. In contrast, IgG B cells sustained higher per-cell ERK1/2 phosphorylation over time. This dichotomy suggested a mechanism for dampening signals transmitted by IgM. Regulatory phosphatase activity in IgM B cells was BCR-mediated and initiated more slowly than kinase activity. This BCR-mediated phosphatase activity was sensitive to inhibition by H2O2 and required to attenuate IgM BCR signaling. These results provide the first kinetic maps of BCR signaling in primary human B cell subsets and enable new studies of signaling in B cell disorders, such as autoimmunity and cancer.
CD8αα-Mediated Intraepithelial Lymphocyte Snatching of Thymic Leukemia MHC Class Ib Molecules In Vitro and In Vivo
Authors: Pardigon Nathalie , Takeda Kazuyo , Saunier Bertrand , Hornung Felicita , Gibbs James , Weisberg Andrea , Contractor Nikhat , Kelsall Brian , Bennink Jack R. , Yewdell Jonathan W.
Published: April 2014

Thymic leukemia (TL) is a MHC class Ib molecule that interacts with CD8αα homodimers. CD8αα is abundantly expressed by intraepithelial T lymphocytes (IELs) located in close proximity to TL-expressing intestinal epithelial cells. In this study, we show that CD8αα+ IELs “snatch” TL from the plasma membrane of TL-expressing cells and express TL in its proper orientation on their own cell surface. TL snatching is enhanced by cross-linking of IEL TCRs in a phosphatidylinositol kinase-dependent manner, and results in overall alterations to the IEL cell surface detected by enhanced binding of peanut agglutinin lectin. Induction of bowel inflammation results in the presence of TL on IELs, probably via in vivo snatching, providing the initial evidence for the interaction of CD8αα IELs with intestinal cells.
New Generation Vaccine Induces Effective Melanoma-Specific CD8+ T Cells in the Circulation but Not in the Tumor Site
Authors: Appay Victor , Jandus Camilla , Voelter Verena , Reynard Severine , Coupland Sarah E. , Rimoldi Donata , Lienard Danielle , Guillaume Philippe , Krieg Arthur M. , Cerottini Jean-Charles , Romero Pedro , Leyvraz Serge , Rufer Nathalie , Speiser Daniel E.
Published: April 2014

Although increasing evidence suggests that CTL are important to fight the development of some cancers, the frequency of detectable tumor-specific T cells is low in cancer patients, and these cells have generally poor functional capacities, compared with virus-specific CD8+ T cells. The generation with a vaccine of potent CTL responses against tumor Ags therefore remains a major challenge. In the present study, ex vivo analyses of Melan-A-specific CD8+ T cells following vaccination with Melan-A peptide and CpG oligodeoxynucleotides revealed the successful induction in the circulation of effective melanoma-specific T cells, i.e., with phenotypic and functional characteristics similar to those of CTL specific for immunodominant viral Ags. Nonetheless, the eventual impact on tumor development in vaccinated melanoma donors remained limited. The comprehensive study of vaccinated patient metastasis shows that vaccine-driven tumor-infiltrating lymphocytes, although activated, still differed in functional capacities compared with blood counterparts. This coincided with a significant increase of FoxP3+ regulatory T cell activity within the tumor. The consistent induction of effective tumor-specific CD8+ T cells in the circulation with a vaccine represents a major achievement; however, clinical benefit may not be achieved unless the tumor environment can be altered to enable CD8+ T cell efficacy.
Filamin A Is Required for T Cell Activation Mediated by Protein Kinase C-θ
Authors: Hayashi Keitaro , Altman Amnon
Published: April 2014

Induction of T cell responses following engagement of the Ag-specific TCR depends on TCR-initiated rearrangements of the cellular actin cytoskeleton and highly coordinated and tightly regulated interactions and of diverse intracellular signaling proteins. In this study, we show that filamin A (FLNa), an actin-binding and signal mediator scaffolding protein, is required for T cell activation. Following Ag stimulation, FLNa was recruited to the T cell-APC contact area, where it colocalized with protein kinase C-θ (PKCθ). Depletion of FLNa by RNA interference did not affect TCR-induced early tyrosine phosphorylation or actin polymerization but, nevertheless, resulted in impaired IL-2 expression by human primary T cells and reduced activation of NF-κB, AP-1, and NFAT reporter genes in transfected T cells. TCR stimulation induced stable physical association of FLNa with PKCθ. Furthermore, the TCR/CD28-induced membrane translocation of PKCθ was inhibited in FLNa-depleted T cells. These results reveal novel role for FLNa in the TCR/CD28 signaling pathway leading to transcription factor activation and IL-2 production, and suggest that this role is mediated, in part, through the inducible interaction of FLNa with PKCθ.
Degalactosylated and/or Denatured IgA, but Not Native IgA in Any Form, Bind to Mannose-Binding Lectin
Authors: Terai Itaru , Kobayashi Kunihiko , Vaerman Jean-Pierre , Mafune Naoki
Published: April 2014

Mannose-binding lectin (MBL) is reported to bind to agalactosyl IgG, but not to normally galactosylated (native) IgG. It was recently reported that serum polymeric IgA in its native form reacts with MBL, whereas a more recent report has claimed that native IgD and IgE, and possibly IgM, do not. This led us to investigate whether IgA is truly reactive with MBL. To accomplish this, we collected purified human Igs, of various classes, subclasses, and allotypes, and tested their ability to bind to MBL using an ELISA method. Among these preparations, only one (monoclonal IgA2m(2):Kur) exhibited significant MBL binding. In particular, polymeric or monomeric forms of our normal serum IgA preparation lacked any ability to bind to MBL whatsoever. However, all the Ig preparations which had not bound to MBL became able to do so when they were degalactosylated with a galactosidase treatment, and the binding was further enhanced by acidic denaturation of the Igs. Among the degalactosylated and/or acid-denatured IgA, the IgA2 subclass exhibited a higher level of MBL binding than did IgA1. Our results suggest that MBL does not bind to native Igs (viewed in principle as “self” components), and that only Igs with abnormal glycosylation (degalactosylated forms) and/or denaturation would be MBL reactive.
Innate Inflammatory Signals Induced by Various Pathogens Differentially Dictate the IFN-I Dependence of CD8 T Cells for Clonal Expansion and Memory Formation
Authors: Thompson Lucas J. , Kolumam Ganesh A. , Thomas Sunil , Murali-Krishna Kaja
Published: April 2014

Type-I IFNs (IFN-I) provide direct survival signals to T cells during Ag-driven proliferation. Because IFN-I production differs depending on the pathogen, we assessed CD8 T cell requirement for direct IFN-I signals during responses to vaccinia virus (VV), vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), and Listeria monocytogenes (LM) immunizations in vivo. IFN-I-receptor-deficient (IFN-IR°) CD8 T cells expanded 3- to 5-fold less and formed a diminished memory pool compared with wild-type (WT) CD8 T cells in response to VV, VSV, or LM. WT CD8 T cells expanded more robustly in response to LCMV-encoded Ags than to Ags encoded by the other three pathogens, and under these conditions the lack of direct IFN-I signals inhibited their expansion by ∼100-fold. To test whether the high antigenic-load provided by LCMV caused greater expansion and greater IFN-I dependency, we primed WT and IFN-IR° OVA-specific OT-1 CD8 T cells with a fixed-number of OVA-peptide-pulsed dendritic cells along with adjuvant effect provided by LCMV, VV, VSV, or LM. Both WT and IFN-IR° OT-1 cells were recruited, proliferated, and differentiated into effectors in all the four cases. However, WT OT-1 cells expanded similarly in all four cases. IFN-IR° OT-1 cells expanded ∼20-fold less than the WT OT-1 CD8 T cells when LCMV was used as adjuvant, whereas their expansion was affected only marginally when VV, VSV, or LM were used as adjuvants. Thus, innate/inflammatory signals induced by different pathogens contribute to CD8 T cell expansion and memory formation via distinct levels of IFN-I dependence.
Allergic Airway Inflammation Inhibits Pulmonary Antibacterial Host Defense
Authors: Beisswenger Christoph , Kandler Kerstin , Hess Christian , Garn Holger , Felgentreff Kerstin , Wegmann Michael , Renz Harald , Vogelmeier Claus , Bals Robert
Published: April 2014

The innate immune system of the lung is a multicomponent host defense system and in addition has an instructing role in regulating the quality and quantity of the adaptive immune response. When the interaction between innate and adaptive immunity is disturbed, pathological conditions such as asthma can develop. It was the aim of the study to investigate the effect of the allergic inflammation of the lung on the innate host defense during bacterial infection. Human bronchial epithelial cells were preincubated with Th2 cytokines and infected with Pseudomonas aeruginosa. The effect of the Th2 cytokines on the mRNA levels of antimicrobial peptides and the antimicrobial activity of HBEC was determined. To investigate the influence of an allergic inflammation on pulmonary host defense in vivo, mice sensitized and challenged with OVA were infected with P. aeruginosa, and the number of viable bacteria in the lungs was determined together with markers of inflammation like cytokines and antimicrobial peptides. Exposure of airway epithelial cells to Th2 cytokines resulted in a significantly decreased antimicrobial activity of the cells and in suppressed mRNA levels of the antimicrobial peptide human β-defensin 2. Furthermore, mice with allergic airway inflammation had significantly more viable bacteria in their lungs after infection. This was consistent with reduced levels of proinflammatory cytokines and of the antimicrobial peptide cathelin-related antimicrobial peptide. These results show that an allergic airway inflammation suppresses the innate antimicrobial host defense. The adaptive immune system modulates the functions of the pulmonary innate immune system.
A Novel 40-kDa Protein Containing Six Repeats of an Epidermal Growth Factor-Like Domain Functions as a Pattern Recognition Protein for Lipopolysaccharide
Authors: Ju Jin Sung , Cho Mi Hyang , Brade Lore , Kim Jung Hyun , Park Ji Won , Ha Nam-Chul , Söderhäll Irene , Söderhäll Kenneth , Brade Helmut , Lee Bok Luel
Published: April 2014

Determination of structures and functions of pattern recognition proteins are important for understanding pathogen recognition mechanisms in host defense and for elucidating the activation mechanism of innate immune reactions. In this study, a novel 40-kDa protein, named LPS recognition protein (LRP), was purified to homogeneity from the cell-free plasma of larvae of the large beetle, Holotrichia diomphalia. LRP exhibited agglutinating activities on Escherichia coli, but not on Staphylococcus aureus and Candida albicans. This E. coli-agglutinating activity was preferentially inhibited by the rough-type LPS with a complete core oligosaccharide. LRP consists of 317 aa residues and six repeats of an epidermal growth factor-like domain. Recombinant LRP expressed in a baculovirus system also showed E. coli agglutination activity in vitro and was able to neutralize LPS by inhibition of LPS-induced IL-6 production in mouse bone marrow mast cells. Furthermore, E. coli coated with the purified LRP were more rapidly cleared in the Holotrichia larvae than only E. coli, indicating that this protein participates in the clearance of E. coli in vivo. The three amino-terminal epidermal growth factor-like domains of LRP, but not the three carboxyl epidermal growth factor-like domains, are involved in the LPS-binding activity. Taken together, this LRP functions as a pattern recognition protein for LPS and plays a role as an innate immune protein.
CXCR3 and IFN Protein-10 inPneumocystisPneumonia
Authors: McAllister Florencia , Ruan Sanbao , Steele Chad , Zheng Mingquan , McKinley Laura , Ulrich Lauren , Marrero Luis , Shellito Judd E. , Kolls Jay K.
Published: April 2014

We have previously shown that Tc1 CD8+ T cells have in vitro and in vivo effector activity against Pneumocystis (PC) infection in mice. Because these cells have preferential expression of CXCR3, we investigated whether CXCR3 was required for host defense activity against PC. Mice deficient in CXCR3 but CD4+ T cell intact, showed an initial delay but were able to clear the infectious challenge, indicating that CXCR3 signaling is not essential for clearance of PC. CD4-depleted mice had lower levels of monokine induced by IFN-γ, IFN protein-10 (IP-10), and IFN-inducible T cell α-chemoattractant at day 7 of infection and are permissive to PC infection. Overexpression of IP-10 in the lungs by adenoviral gene transfer did not accelerate clearance of infection in control mice but accelerated clearance by day 28 in mice depleted of CD4+ T cells. This effect was associated with increased recruitment of CD8+ T to the lungs with higher CXCR3+ expression levels and enhanced IFN-γ secretion upon in vitro activation compared with control mice. These results indicate that the CXCR3 chemokines are part of the host defense response to PC, and that IP-10 can direct Tc1 CD8+ T cell recruitment to the lungs and contribute to host defense against PC even in the absence of CD4+ T cells.
Both CXCR3 and CXCL10/IFN-Inducible Protein 10 Are Required for Resistance to Primary Infection by Dengue Virus
Authors: Hsieh Ming-Fang , Lai Szu-Liang , Chen Jia-Perng , Sung Jui-Ming , Lin Yi-Ling , Wu-Hsieh Betty A. , Gerard Craig , Luster Andrew , Liao Fang
Published: April 2014

We examined the extent to which CXCR3 mediates resistance to dengue infection. Following intracerebral infection with dengue virus, CXCR3-deficient (CXCR3−/−) mice showed significantly higher mortality rates than wild-type (WT) mice; moreover, surviving CXCR3−/− mice, but not WT mice, often developed severe hind-limb paralysis. The brains of CXCR3−/− mice showed higher viral loads than those of WT mice, and quantitative analysis using real-time PCR, flow cytometry, and immunohistochemistry revealed fewer T cells, CD8+ T cells in particular, in the brains of CXCR3−/− mice. This suggests that recruitment of effector T cells to sites of dengue infection was diminished in CXCR3−/− mice, which impaired elimination of the virus from the brain and thus increased the likelihood of paralysis and/or death. These results indicate that CXCR3 plays a protective rather than an immunopathological role in dengue virus infection. In studies to identify critical CXCR3 ligands, CXCL10/IFN-inducible protein 10-deficient (CXCL10/IP-10−/−) mice infected with dengue virus showed a higher mortality rate than that of the CXCR3−/− mice. Although CXCL10/IP-10, CXCL9/monokine induced by IFN-γ, and CXCL11/IFN-inducible T cell α chemoattractant share a single receptor and all three of these chemokines are induced by dengue virus infection, the latter two could not compensate for the absence of CXCL10/IP-10 in this in vivo model. Our results suggest that both CXCR3 and CXCL10/IP-10 contribute to resistance against primary dengue virus infection and that chemokines that are indistinguishable in in vitro assays differ in their activities in vivo.