bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for NK Cell Subsets Based on Chemokine Receptor Expression
Authors: Berahovich Robert D. , Lai Nu L. , Wei Zheng , Lanier Lewis L. , Schall Thomas J.
Published: April 2014

To help understand the role of chemokines in NK cell trafficking, we determined the chemokine receptor profiles of three different human NK cell lines and freshly isolated primary human NK cells. The cell lines overlapped in their chemokine receptor profiles: CXCR3 and CXCR4 were expressed by all three lines, whereas CCR1, CCR4, CCR6, CCR7, and CX3CR1 were expressed by only one or two of the lines, and no other chemokine receptors were detected. Freshly isolated primary NK cells were found to express CXCR1, CXCR3, and CXCR4, and to contain subsets expressing CCR1, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR5, and CXCR6. With the exception of CCR4, these chemokine receptors were expressed at higher percentages by CD56bright NK cells than by CD56dim NK cells. In particular, CCR7 was expressed by almost all CD56bright NK cells but was not detected on CD56dim NK cells. CCR9 and CXCR6 have not been described previously on primary NK cells. These results indicate that within both the CD56bright and CD56dim NK cell populations, subsets with the capacity for differential trafficking programs exist, which likely influence their functions in innate and adaptive immunity.
Mapping of Quantitative Trait Loci Determining NK Cell-Mediated Resistance to MHC Class I-Deficient Bone Marrow Grafts in Perforin-Deficient Mice
Authors: Johansson Maria H. , Taylor Mesha A. , Jagodic Maja , Tus Katalin , Schatzle John D. , Wakeland Edward K. , Bennett Michael
Published: April 2014

NK cells reject allogeneic and MHC class I-deficient bone marrow (BM) grafts in vivo. The mechanisms used by NK cells to mediate this rejection are not yet thoroughly characterized. Although perforin plays a major role, perforin-independent mechanisms are involved as well. C57BL/6 mice deficient in perforin (B6 perforin knockout (PKO)) reject class I-deficient TAP-1 KO BM cells as efficiently as normal B6 mice. In contrast, perforin-deficient 129S6/SvEvTac mice (129 PKO) cannot mediate this rejection while normal 129 mice efficiently reject. This suggests that in 129, but not in B6, mice, perforin is crucial for NK cell-mediated rejection of MHC class I-deficient BM grafts. To identify loci linked to BM rejection in perforin-deficient mice, we generated backcross 1 progeny by crossing (129 × B6)F1 PKO mice to 129 PKO mice. In transplantation experiments, >350 backcross 1 progeny were analyzed and displayed a great variation in ability to reject TAP-1 KO BM grafts. PCR-based microsatellite mapping identified four quantitative trait loci (QTL) on chromosomes 2, 4, and 8, with the QTL on chromosome 8 showing the highest significance, as well as a fifth epistatic QTL on chromosome 3. This study describes the first important step toward identifying BM graft resistance gene(s).
Increased Natural CD4+CD25+ Regulatory T Cells and Their Suppressor Activity Do Not Contribute to Mortality in Murine Polymicrobial Sepsis
Authors: Scumpia Philip O. , Delano Matthew J. , Kelly Kindra M. , O’Malley Kerri A. , Efron Philip A. , McAuliffe Priscilla F. , Brusko Todd , Ungaro Ricardo , Barker Tolga , Wynn James L. , Atkinson Mark A. , Reeves Westley H. , Clare Salzler Michael J. , Moldawer Lyle L.
Published: April 2014

Regulatory T cells (Tregs), including natural CD4+CD25+ Tregs and inducible IL-10 producing T regulatory type 1 (TR1) cells, maintain tolerance and inhibit autoimmunity. Recently, increased percentages of Tregs have been observed in the blood of septic patients, and ex vivo-activated Tregs were shown to prevent polymicrobial sepsis mortality. Whether endogenous Tregs contribute to sepsis outcome remains unclear. Polymicrobial sepsis, induced by cecal ligation and puncture, caused an increased number of splenic Tregs compared with sham-treated mice. Splenic CD4+CD25+ T cells from septic mice expressed higher levels of Foxp3 mRNA and were more efficient suppressors of CD4+CD25− T effector cell proliferation. Isolated CD4+ T cells from septic mice displayed increased intracellular IL-10 staining following stimulation, indicating that TR1 cells may also be elevated in sepsis. Surprisingly, Ab depletion of total CD4+ or CD4+CD25+ populations did not affect mortality. Furthermore, no difference in survival outcome was found between CD25 or IL-10 null mice and wild-type littermates, indicating that Treg or TR1-generated IL-10 are not required for survival. These results demonstrate that, although sepsis causes a relative increase in Treg number and increases their suppressive function, their presence does not contribute significantly to overall survival in this model.
Neutrophils Are a Key Component of the Antitumor Efficacy of Topical Chemotherapy with Ingenol-3-Angelate
Authors: Challacombe Jodie M. , Suhrbier Andreas , Parsons Peter G. , Jones Brad , Hampson Peter , Kavanagh Dean , Rainger G. Ed , Morris Melanie , Lord Janet M. , Le Thuy T. T. , Hoang-Le Diem , Ogbourne Steven M.
Published: April 2014

Harnessing neutrophils for the eradication of cancer cells remains an attractive but still controversial notion. In this study, we provide evidence that neutrophils are required to prevent relapse of skin tumors following topical treatment with a new anticancer agent, ingenol-3-angelate (PEP005). Topical PEP005 treatment induces primary necrosis of tumor cells, potently activates protein kinase C, and was associated with an acute T cell-independent inflammatory response characterized by a pronounced neutrophil infiltrate. In Foxn1nu mice depleted of neutrophils and in CD18-deficient mice (in which neutrophil extravasation is severely impaired) PEP005 treatment was associated with a >70% increase in tumor relapse rates. NK cell or monocyte/macrophage deficiency had no effect on relapse rates. Both in vitro and in mice, PEP005 induced MIP-2/IL-8, TNF-α, and IL-1β, all mediators of neutrophil recruitment and activation. In vitro, PEP005 activated human endothelial cells resulting in neutrophil adhesion and also induced human neutrophils to generate tumoricidal-reactive oxygen intermediates. Treatment of tumors with PEP005 significantly elevated the level of anticancer Abs, which were able to promote neutrophil-mediated Ab-dependent cellular cytotoxicity (ADCC) in vitro. PEP005 treatment of tumors grown in SCID mice was also associated with >70% increase in tumor relapse rates. Taken together, these data suggest a central role for neutrophil-mediated ADCC in preventing relapse. PEP005-mediated cure of tumors therefore appears to involve initial chemoablation followed by a neutrophil-dependent ADCC-mediated eradication of residual disease, illustrating that neutrophils can be induced to mediate important anticancer activity with specific chemotherapeutic agents.
Cutting Edge: TCR-Induced NAB2 Enhances T Cell Function by Coactivating IL-2 Transcription
Authors: Collins Samuel , Wolfraim Lawrence A. , Drake Charles G. , Horton Maureen R. , Powell Jonathan D.
Published: April 2014

TCR engagement leads to the up-regulation of genetic programs that can both activate and inhibit T cell function. The early growth receptor (Egr) proteins Egr-2 and Egr-3 have recently been identified as TCR-induced negative regulators of T cell function. NAB2 (NGFI-A-binding protein 2) is both a coactivator and a corepressor of Egr-mediated transcription and has been implicated in regulating Schwann cell myelination. In this report we demonstrate that NAB2 is induced by TCR engagement and that its expression is enhanced by the presence of costimulation. The overexpression of NAB2 enhanced IL-2 production while small interfering RNA to NAB2 markedly inhibited IL-2 expression. Mechanistically, we demonstrate that NAB2 enhances IL-2 transcription by acting as a coactivator for Egr-1. Indeed, chromatin immunoprecipitation analysis reveals that NAB2 is recruited to the Egr-1 binding site of the IL-2 promoter. Taken together, our findings identify NAB2 as a novel coactivator of T cell function.
Human Mast Cell Chymase Cleaves Pro-IL-18 and Generates a Novel and Biologically Active IL-18 Fragment
Authors: Omoto Youichi , Tokime Kazuya , Yamanaka Keiichi , Habe Koji , Morioka Tatsuhiko , Kurokawa Ichiro , Tsutsui Hiroko , Yamanishi Kiyofumi , Nakanishi Kenji , Mizutani Hitoshi
Published: April 2014

Increased release of IL-18 in the skin causes atopic dermatitis (AD)-like skin lesions, suggesting a role of IL-18 in the pathogenesis of AD. Caspase-1 is a well-known activator of IL-18, but caspase-1 knockout mice still have biologically active IL-18. Normal human keratinocyte constitutively produces pro-IL-18, but it is unable to activate it, suggesting the existence of an alternative pathway for IL-18 in the skin. Dermal accumulation of mast cells is commonly observed in AD patients and in experimental mouse models of AD. Connective tissue mast cells contain high amounts of chymase and tryptase in their cytoplasmic granules. In the present study, we demonstrated that activation of IL-18 is a novel function of human mast cell chymase. Human mast cell chymase rapidly cleaves recombinant pro-IL-18 at 56-phenylalanine and produces a biologically active IL-18 fragment that is smaller than any other reported IL-18-derived species. The human mast cell chymase and the novel IL-18-derived active peptide may be novel therapeutic targets in AD- and IL-18-associated diseases
Severely Impaired Clonal Deletion of CD4+ T Cells in Low-Dose Irradiated Mice: Role of T Cell Antigen Receptor and IL-7 Receptor Signals
Authors: Shklovskaya Elena , Fazekas de St. Groth Barbara
Published: April 2014

Systemic administration of high doses of soluble Ag induces peripheral CD4+ T cell tolerance in unmanipulated hosts. To test whether tolerance is modified under conditions of transient lymphopenia, we tracked the response of 5C.C7 TCR-transgenic CD4+ T cells to i.v. moth cytochrome c peptide in mice that received low-dose gamma irradiation 10 days previously. This model was chosen because it does not support spontaneous lymphopenia-induced proliferation of 5C.C7 cells, allowing the study of Ag-specific responses without interference from simultaneous spontaneous proliferation. Clonal expansion in response to i.v. peptide was increased in irradiated mice, while clonal deletion was severely impaired in comparison with untreated animals. Amplified TCR triggering was observed in irradiated hosts, consistent with dendritic cell activation leading to enhanced Ag presentation. Failure of deletion was accompanied by persistent T cell activation and accumulation of Th1 effector cells. Up-regulated expression of IL-7R and the prosurvival protein Bcl-xL was associated with clonal persistence. Cells with memory and naive phenotypes were both represented within persistent clones, but no Th1 function could be demonstrated within the long-term memory population. Failure of clonal deletion in irradiated hosts represents a novel mechanism limiting TCR diversity in a lymphopenic environment and may contribute to subsequent autoimmunity.
Latent Virus Influences the Generation and Maintenance of CD8+ T Cell Memory
Authors: Sheridan Brian S. , Khanna Kamal M. , Frank Gregory M. , Hendricks Robert L.
Published: April 2014

The influence of latent virus on CD8+ T cell memory is poorly understood. HSV type 1 specifically establishes latency in trigeminal ganglia (TG) after corneal infection of mice. In latently infected TG, IL-15 deprivation reduced the following: 1) accumulation of HSV-specific CD8+ effector T cells (HSV-CD8eff), 2) accumulation of CD127+ putative HSV-CD8 memory precursors, and 3) the size and functionality of the memory (HSV-CD8mem) population. Although compromised in IL-15−/− mice, the HSV-CD8mem pool persisted in latently infected tissue, but not in noninfected tissue of the same mice. Anti-IL-2 treatment also dramatically reduced the size of the HSV-CD8eff population in the TG, but did not influence the concomitant generation of the CD127+ putative HSV-CD8mem precursor population or the size or functionality of the HSV-CD8mem pool. Thus, the size of the memory pool appears to be determined by the size of the CD127+ CD8mem precursor population and not by the size of the overall CD8eff pool. HSV-CD8mem showed a higher basal rate of proliferation in latently infected than noninfected tissue, which was associated with a reduced population of CD4+FoxP3+ regulatory T cells. Thus, the generation, maintenance, and function of memory CD8+ T cells is markedly influenced by latent virus.
Heat Shock Up-Regulates lmp2 and lmp7 and Enhances Presentation of Immunoproteasome-Dependent Epitopes
Authors: Callahan Margaret K. , Wohlfert Elizabeth A. , Ménoret Antoine , Srivastava Pramod K.
Published: April 2014

The heat shock response is a canonical regulatory pathway by which cellular stressors such as heat and oxidative stress alter the expression of stress-responsive genes. Some of these stress-responsive genes (heat shock proteins and MHC class I (MHC I)-related chains) play a significant role in the immune system. In this study, we have investigated the impact of stimulating the heat shock response on genes involved in the MHC I presentation pathway. We report that two inducible subunits of the proteasome, lmp2 and lmp7, are transcriptionally up-regulated by heat shock in cells of mouse and human origin. Furthermore, heat-shocked cells show enhanced presentation of the immunoproteasome-dependent MHC I antigenic epitopes NP118–126 of lymphocytic choriomeningitis virus and E1B192–200 of adenovirus, but not immunoproteasome-independent epitopes such as tumor Ag AH1 and SV40 large T Ag epitope II223–231. These findings show a novel immunological sequel to the cellular response to stress that may play a key role during fever or other homeostatic perturbations.
Semimature Stage: A Checkpoint in a Dendritic Cell Maturation Program That Allows for Functional Reversion after Signal-Regulatory Protein-α Ligation and Maturation Signals
Authors: Braun Deborah , Galibert Laurent , Nakajima Toshiharu , Saito Hirohisa , Quang Van Vu , Rubio Manuel , Sarfati Marika
Published: April 2014

CD47 on live cells actively engages signal-regulatory protein-α (SIRP-α) on phagocytes and delivers a negative signal that prevents their elimination. We evaluated the biological consequences of SIRP-α ligation on the dendritic cell (DC) response to maturation signals and the potential interplay with the IL-10/IL-10R inhibitory pathway. At first, CD47/SIRP-α allowed the generation of mature migratory DCs not producing IL-12, IFN-γ-inducible protein-10, and CCL19. Rather, they secreted neutrophils attracting chemokine CXCL5 and IL-1β, reflecting a partial block in functional DC maturation. Afterward, semimature DCs functionally regressed in an IL-10-independent fashion toward cells that retrieved the cardinal features of immature DCs: re-expression of CCR5, loss of DC-lysosome-associated membrane protein, high endocytosis, and impaired allostimulatory functions. The global gene expression profile of IL-10 and SIRP-α-ligated DC demonstrated two distinct molecular pathways. IL-10R and SIRP-α expression were reciprocally down-regulated by CD47 and IL-10, respectively. These results emphasize that the SIRP-α pathway might be part of the molecular machinery used by the DC to dampen or resolve an inflammatory response in an IL-10-independent manner.
Molecular Basis of TCR Selectivity, Cross-Reactivity, and Allelic Discrimination by a Bacterial Superantigen: Integrative Functional and Energetic Mapping of the SpeC-Vβ2.1 Molecular Interface
Authors: Rahman A. K. M. Nur-ur , Herfst Christine A. , Moza Beenu , Shames Stephanie R. , Chau Luan A. , Bueno Clara , Madrenas Joaquín , Sundberg Eric J. , McCormick John K.
Published: April 2014

Superantigens activate large fractions of T cells through unconventional interactions with both TCR β-chain V domains (Vβs) and MHC class II molecules. The bacterial superantigen streptococcal pyrogenic exotoxin C (SpeC) primarily stimulates human Vβ2+ T cells. Herein, we have analyzed the SpeC-Vβ2.1 interaction by mutating all SpeC residues that make contact with Vβ2.1 and have determined the energetic and functional consequences of these mutations. Our comprehensive approach, including mutagenesis, functional readouts from both bulk T cell populations, and an engineered Vβ2.1+ Jurkat T cell, as well as surface plasmon resonance binding analysis, has defined the SpeC “functional epitope” for TCR engagement. Although only two SpeC residues (Tyr15 and Arg181) are critical for activation of virtually all human CD3+ T cells, a larger cluster of four hot spot residues are required for interaction with Vβ2.1. Three of these residues (Tyr15, Phe75, and Arg181) concentrate their binding energy on the CDR2 loop residue Ser52a, a noncanonical residue insertion found only in Vβ2 and Vβ4 chains. Plasticity of this loop is important for recognition by SpeC. Although SpeC interacts with the Vβ2.1 hypervariable CDR3 loop, our data indicate these contacts have little to no influence on the functional interaction with Vβ2.1. These studies also provide a molecular basis for selectivity and cross-reactivity of SpeC-TCR recognition and reveal a degree of fine specificity in these interactions, whereby certain SpeC mutants are capable of distinguishing between different alleles of the same Vβ domain subfamily.
NAK-Associated Protein 1 Participates in Both the TLR3 and the Cytoplasmic Pathways in Type I IFN Induction
Authors: Sasai Miwa , Shingai Masashi , Funami Kenji , Yoneyama Mitsutoshi , Fujita Takashi , Matsumoto Misako , Seya Tsukasa
Published: April 2014

TLR3 and the cytoplasmic helicase family proteins (retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5)) serve as dsRNA pattern-recognition receptors. In response to poly(I:C), a representative of dsRNA, and viral infection, they have been shown to activate the transcription factor IFN regulatory factor (IRF)-3, which in turn induces activation of the IFN-β promoter. RIG-I/MDA5 recognizes dsRNA in the cytoplasm, whereas TLR3 resides in the cell surface membrane or endosomes to engage in extracytoplasmic recognition of dsRNA. Recent reports suggest that TLR3 induces cellular responses in epithelial cells in response to respiratory syncytial virus (RSV). The modus for TLR3 activation by RSV, however, remains unresolved. By small interference RNA gene-silencing technology and human cell transfectants, we have revealed that knockdown of NAK-associated protein 1 (NAP1) leads to the down-regulation of IFN-β promoter activation >24 h after poly(I:C) or virus (RSV and vesicular stomatitis virus) treatment. NAP1 is located downstream of the adapter Toll-IL-1R homology domain-containing adapter molecule (TICAM)-1 (Toll/IL-1R domain-containing adapter-inducing IFN-β) in the TLR3 pathway, but TICAM-1 and TLR3 did not participate in the IRF-3 and IFN-β promoter activation by RSV infection. Virus-mediated activation of the IFN-β promoter was largely abrogated by the gene silencing of IFN-β promoter stimulator-1 (mitochondria antiviral signaling (MAVS), VISA, Cardif), the adapter of the RIG-I/MDA5 dsRNA-recognition proteins. In both the TLR and virus-mediated IFN-inducing pathways, IκB kinase-related kinase ε and TANK-binding kinase 1 participated in IFN-β induction. Thus, RSV as well as other viruses induces replication-mediated activation of the IFN-β promoter, which is intracellularly initiated by the RIG-I/MDA5 but not the TLR3 pathway. Both the cytoplasmic and TLR3-mediated dsRNA recognition pathways converge upon NAP1 for the activation of the IRF-3 and IFN-β promoter.
High Mobility Group B1 Protein Suppresses the Human Plasmacytoid Dendritic Cell Response to TLR9 Agonists
Authors: Popovic Petar J. , DeMarco Richard , Lotze Michael T. , Winikoff Steven E. , Bartlett David L. , Krieg Arthur M. , Guo Z. Sheng , Brown Charles K. , Tracey Kevin J. , Zeh Herbert J.
Published: April 2014

Plasmacytoid dendritic cells (PDC) are innate immune effector cells that are recruited to sites of chronic inflammation, where they modify the quality and nature of the adaptive immune response. PDCs modulate adaptive immunity in response to signals delivered within the local inflammatory milieu by pathogen- or damage-associated molecular pattern, molecules, and activated immune cells (including NK, T, and myeloid dendritic cells). High mobility group B1 (HMGB1) is a recently identified damage-associated molecular pattern that is released during necrotic cell death and also secreted from activated macrophages, NK cells, and mature myeloid dendritic cells. We have investigated the effect of HMGB1 on the function of PDCs. In this study, we demonstrate that HMGB1 suppresses PDC cytokine secretion and maturation in response to TLR9 agonists including the hypomethylated oligodeoxynucleotide CpG- and DNA-containing viruses. HMGB1-inhibited secretion of several proinflammatory cytokines including IFN-α, IL-6, TNF-α, inducible protein-10, and IL-12. In addition, HMGB1 prevented the CpG induced up-regulation of costimulatory molecules on the surface of PDC and potently suppressed their ability to drive generation of IFN-γ-secreting T cells. Our observations suggest that HMGB1 may play a critical role in regulating the immune response during chronic inflammation and tissue damage through modulation of PDC function.
Thiocyanate-Dependent Induction of Endothelial Cell Adhesion Molecule Expression by Phagocyte Peroxidases: A Novel HOSCN-Specific Oxidant Mechanism to Amplify Inflammation
Authors: Wang Jian-Guo , Mahmud Shawn A. , Nguyen Julia , Slungaard Arne
Published: April 2014

Both eosinophil peroxidase (EPO) and neutrophil myeloperoxidase (MPO) preferentially oxidize SCN− to generate HOSCN, a weak, sulfhydryl-reactive oxidant, as a major physiologic product. We here show that HOSCN is a uniquely potent phagocyte oxidant inducer of E-selectin, ICAM-1, and VCAM-1 expression in HUVEC as detected by Western blot and flow cytometry. EMSA and inhibitor studies show that HOSCN up-regulation of these adhesion molecules is transcriptionally mediated through a mechanism that is dependent upon activation of the NF-κB p65/p50 transcription factor and constitutively suppressed by PI3K-Akt pathway activity. HUVEC monolayers exposed to HOSCN bind 8-fold more neutrophils and 3- to 4-fold more Aml14.3D10 cells (a differentiated cell line model of mature eosinophils) than control monolayers. Blocking Ab studies confirm the involvement of E-selectin and ICAM-1 but not VCAM-1 in neutrophil adhesion and of all three in Aml14.3D10 adhesion. Intraperitoneal injection of HOSCN evoked an 8-fold increase in neutrophil peritoneal extravasation. In addition to NF-κB, HOSCN also activates the potentially proinflammatory transcription factors Stat4, CDP, GRE, CBF, Ets-1/PEA3, and TFIID, a pattern easily distinguishable from that induced by LPS. These results suggest that phagocyte peroxidases function to amplify inflammation through a novel, HOSCN-specific oxidant mechanism.
Induction of Vascular Leakage and Blood Pressure Lowering through Kinin Release by a Serine Proteinase from Aeromonas sobria
Authors: Imamura Takahisa , Kobayashi Hidemoto , Khan Rasel , Nitta Hidetoshi , Okamoto Keinosuke
Published: April 2014

Aeromonas sobria causes septic shock, a condition associated with high mortality. To study the mechanism of septic shock by A. sobria infection, we examined the vascular leakage (VL) activity of A. sobria serine proteinase (ASP), a serine proteinase secreted by this pathogen. Proteolytically active ASP induced VL mainly in a bradykinin (BK) B2 receptor-, and partially in a histamine-H1 receptor-dependent manner in guinea pig skin. The ASP VL activity peaked at 10 min to 1.8-fold of the initial activity with an increased BK B2 receptor dependency, and attenuated almost completely within 30 min. ASP produced VL activity from human plasma apparently through kallikrein/kinin system activation, suggesting that ASP can generate kinin in humans. Consistent with the finding that a major part of the ASP-induced VL was reduced by a potent kallikrein inhibitor, soybean trypsin inhibitor that does not affect ASP enzymatic activity, ASP activated prekallikrein but not factor XII to generate kallikrein in a dose- and incubation time-dependent manner. ASP produced more VL activity directly from human low m.w. kininogen than high m.w. kininogen when both were used at their normal plasma concentrations. Intra-arterial injection of ASP into guinea pigs lowered blood pressure specifically via the BK B2 receptor. These data suggest that ASP induces VL through prekallikrein activation and direct kinin release from kininogens, which is a previously undescribed mechanism of A. sobria virulence and could be associated with the induction of septic shock by infection with this bacterium. ASP-specific inhibitors, and kinin receptor antagonists, might prove useful for the treatment or prevention of this fatal disease.
Oncostatin M-Induced IL-6 Expression in Murine Fibroblasts Requires the Activation of Protein Kinase Cδ
Authors: Smyth David C. , Kerr Christine , Richards Carl D.
Published: April 2014

Oncostatin M (OSM) is an IL-6/LIF cytokine family member whose role has been identified in a range of biological activities in vitro, including up-regulation of inflammatory gene expression and regulation of connective tissue metabolism. However, the mechanisms through which OSM regulates cellular responses are not completely understood. In this study, we show that activation of the calcium-independent or novel protein kinase C (PKC) isoform PKCδ is a critical event during OSM-mediated up-regulation of IL-6 expression in murine fibroblasts. The pan-PKC inhibitor GF109203X (bisindolylmaleimide I) reduced secretion of IL-6; however, use of Go6976, an inhibitor of calcium-dependent PKC enzymes, did not. The PKCδ-selective inhibitory compound rottlerin abrogated expression of IL-6 transcript and protein, but only reduced PKCδ activity when used at higher concentrations as determined by kinase activity assay, suggesting rottlerin may inhibit IL-6 expression in a PKCδ-independent manner. However, silencing of PKCδ protein expression, but not the related novel isoform PKCε, by use of RNA interference (i.e., small interfering RNA) demonstrated that PKCδ is required for murine OSM (mOSM) induction of IL-6 protein secretion. Furthermore, inhibition of PI3K by use of LY294002 reduces expression of IL-6 at both the mRNA and protein level in murine fibroblasts, and we suggest that PI3K is required for activation of PKCδ. Knockdown of phosphoinositide-dependent kinases PDK-1 or Akt1 using small interfering RNA strategies did not influence mOSM-induced IL-6 expression, suggesting mOSM uses a PI3K–PKCδ pathway of activation independent of these kinases. Our findings illustrate a novel signaling network used by mOSM that may be important for its mediation of inflammatory processes.
Cyclooxygenase Regulates Cell Surface Expression of CXCR3/1-Storing Granules in Human CD4+ T Cells
Authors: Gasser Olivier , Schmid Thomas A. , Zenhaeusern Gabriela , Hess Christoph
Published: April 2014

Efficient migration of CD4+ T cells into sites of infection/inflammation is a prerequisite to protective immunity. Inappropriate recruitment, on the other hand, contributes to inflammatory pathologies. The chemokine/chemokine receptor system is thought to orchestrate T cell homing. In this study, we show that most circulating human CD4+ T cells store the inflammatory chemokine receptors CXCR3 and CXCR1 within a distinct intracellular compartment. Equipped with such storage granules, CD4+ T cells coexpressing both receptors increased from only 1% ex vivo to ∼30% within minutes of activation with PHA or exposure to the cyclooxygenase (COX) substrate arachidonic acid. Up-regulation was TCR independent and reduced by COX inhibitors at concentrations readily reached in vivo. The inducible inflammatory CXCR3highCXCR1+ phenotype identified nonpolarized cells, was preferentially triggered on CCR7+CD4+ T cells, and conferred increased chemotactic responsiveness. Thus, inducible CXCR3/1 expression occurs in a large fraction of CD4+ T cells. Its dependency on COX may explain a number of established, and point toward novel, effects of COX inhibitors.
Costimulation via CD55 on Human CD4+ T Cells Mediated by CD97
Authors: Capasso Melania , Durrant Lindy G. , Stacey Martin , Gordon Siamon , Ramage Judith , Spendlove Ian
Published: April 2014

Decay-accelerating factor (CD55) is a complement regulatory protein, which is expressed by most cells to protect them from complement-mediated attack. CD55 also binds CD97, an EGF-TM7 receptor constitutively expressed on granulocytes and monocytes and rapidly up-regulated on T and B cells upon activation. Early results suggested that CD55 could further enhance T cell proliferation induced by phorbol ester treatment. The present study demonstrates that coengagement of CD55, using either cross-linking mAbs or its natural ligand CD97, and CD3 results in enhanced proliferation of human peripheral blood CD4+ T cells, expression of the activation markers CD69 and CD25, and secretion of IL-10 and GM-CSF. Recently, an increase in T cell responsiveness in CD55−/− mice was shown to be mediated by a lack of complement regulation. In this study, we show that direct stimulation of CD55 on CD4+ T cells with CD97 can modulate T cell activation but does not interfere with CD55-mediated complement regulation. Our results support a multifaceted role for CD55 in human T cell activation, constituting a further link between innate and adaptive immunity.
Paucity of V-D-D-J Rearrangements and VH Replacement Events in Lupus Prone and Nonautoimmune TdT−/− and TdT+/+ Mice
Authors: Watson Lisa C. , Moffatt-Blue Chantelle S. , McDonald R. Zachary , Kompfner Elizabeth , Ait-Azzouzene Djemel , Nemazee David , Theofilopoulos Argyrios N. , Kono Dwight H. , Feeney Ann J.
Published: April 2014

CDR3 regions containing two D segments, or containing the footprints of VH replacement events, have been reported in both mice and humans. However, the 12–23 bp rule for V(D)J recombination predicts that D-D rearrangements, which would occur between 2 recombination signal sequences (RSSs) with 12-bp spacers, should be extremely disfavored, and the cryptic RSS used for VH replacement is very inefficient. We have previously shown that newborn mice, which lack TdT due to the late onset of its expression, do not contain any CDR3 with D-D rearrangements. In the present study, we test our hypothesis that most D-D rearrangements are due to fortuitous matching of the second apparent D segment by TdT-introduced N nucleotides. We analyzed 518 sequences from adult MRL/lpr- and C57BL/6 TdT-deficient B cell precursors and found only two examples of CDR3 with D-D rearrangements and one example of a potential VH replacement event. We examined rearrangements from pre-B cells, marginal zone B cells, and follicular B cells from mice congenic for the Lbw5 (Sle3/5) lupus susceptibility loci and from other strains of mice and found very few examples of CDR3 with D-D rearrangements. We assayed B progenitor cells, and cells enriched for receptor editing, for DNA breaks at the “cryptic heptamer” but such breaks were rare. We conclude that many examples of apparent D-D rearrangements in the mouse are likely due to N additions that fortuitously match short stretches of D genes and that D-D rearrangements and VH replacement are rare occurrences in the mouse.
The Histone Methyltransferase Suv39h1 Increases Class Switch Recombination Specifically to IgA
Authors: Bradley Sean P. , Kaminski Denise A. , Peters Antoine H. F. M. , Jenuwein Thomas , Stavnezer Janet
Published: April 2014

Ab class (isotype) switching allows the humoral immune system to adaptively respond to different infectious organisms. Isotype switching occurs by intrachromosomal DNA recombination between switch (S) region sequences associated with CH region genes. Although isotype-specific transcription of unrearranged (germline) CH genes is required for switching, recent results suggest that isotype specificity is also determined by the sequences of downstream (acceptor) S regions. In the current study, we identify the histone methyltransferase Suv39h1 as a novel Sα-specific factor that specifically increases IgA switching (Sμ-Sα recombination) in a transiently transfected plasmid S substrate, and demonstrate that this effect requires the histone methyltransferase activity of Suv39h1. Additionally, B cells from Suv39h1-deficient mice have an isotype-specific reduction in IgA switching with no effect on the level of germline Iα-Cα transcripts. Taken together, our results suggest that Suv39h1 activity inhibits the activity of a sequence-specific DNA-binding protein that represses switch recombination to IgA.
Resistance of C57BL/6 Mice to Amoebiasis Is Mediated by Nonhemopoietic Cells but Requires Hemopoietic IL-10 Production
Authors: Hamano Shinjiro , Asgharpour Amon , Stroup Suzanne E. , Wynn Thomas A. , Leiter Edward H. , Houpt Eric
Published: April 2014

Resistance to intestinal amoebiasis is mouse strain dependent. C57BL/6 (B6) mice clear Entamoeba histolytica within hours of challenge, whereas C3H and CBA strains are susceptible to infection and disease. In this study, we show using bone marrow (BM) chimeric mice that mouse strain-dependent resistance is mediated by nonhemopoietic cells; specifically, B6 BM → CBA recipients remained susceptible as measured by amoeba score and culture, whereas CBA BM → B6 recipients remained resistant. Interestingly, hemopoietic IL-10 was required for maintaining the resistance of B6 mice, in that B6 IL-10-deficient mice and IL-10−/− BM → wild-type recipients, but not IL-10+/+ BM → IL-10−/− recipients, exhibited higher amoeba scores than their wild-type controls. Additionally, C57BL/10 IL-10−/−Rag2−/− mice exhibited diminished amoeba scores and culture rates vs IL-10−/− mice, indicating that lymphocytes potentiated the susceptibility of IL-10-deficient mice. We conclude that nonhemopoietic cells mediate the natural resistance to intestinal amoebiasis of B6 mice, yet this resistance depends on hemopoietic IL-10 activity.
Wolbachia Endosymbiotic Bacteria of Brugia malayi Mediate Macrophage Tolerance to TLR- and CD40-Specific Stimuli in a MyD88/TLR2-Dependent Manner
Authors: Turner Joseph D. , Langley R. Stuart , Johnston Kelly L. , Egerton Gill , Wanji Samuel , Taylor Mark J.
Published: April 2014

Lymphatic filarial nematodes are able to down-regulate parasite-specific and nonspecific responses of lymphocytes and APC. Lymphatic filariae are reliant on Wolbachia endosymbiotic bacteria for development and survival. We tested the hypothesis that repeated exposure to Wolbachia endosymbionts would drive macrophage tolerance in vitro and in vivo. We pre-exposed murine peritoneal-elicited macrophages to soluble extracts of Brugia malayi female worms (BMFE) before restimulating with BMFE or TLR agonists. BMFE tolerized macrophages (in terms of IFN-β, IL-1β, IL-6, IL-12p40, and TNF-α inflammatory cytokine production) in a dose-dependent manner toward self, LPS, MyD88-dependent TLR2 or TLR9 ligands (peptidoglycan, triacyl lipopeptide, CpG DNA) and the MyD88-independent/TRIF-dependent TLR3 ligand, polyinosinic-polycytidylic acid. This was accompanied with down-regulation in surface expression of TLR4 and up-regulation of CD14, CD40, and TLR2. BMFE tolerance extended to CD40 activation in vitro and systemic inflammation following lethal challenge in an in vivo model of endotoxin shock. The mechanism of BMFE-mediated macrophage tolerance was dependent on MyD88 and TLR2 but not TLR4. Evidence that desensitization was driven by Wolbachia-specific ligands was determined by use of extracts from Wolbachia-depleted B. malayi, aposymbiotic filarial species, and a cell line stably infected with Wolbachia pipientis. Our data promote a role for Wolbachia in contributing toward the dysregulated and tolerized immunological phenotype that accompanies the majority of human filarial infections.
A Novel Approach to Characterize Clonality and Differentiation of Human Melanoma-Specific T Cell Responses: Spontaneous Priming and Efficient Boosting by Vaccination
Authors: Speiser Daniel E. , Baumgaertner Petra , Barbey Catherine , Rubio-Godoy Verena , Moulin Alexandre , Corthesy Patricia , Devevre Estelle , Dietrich Pierre-Yves , Rimoldi Donata , Liénard Danielle , Cerottini Jean-Charles , Romero Pedro , Rufer Nathalie
Published: April 2014

Despite major progress in T lymphocyte analysis in melanoma patients, TCR repertoire selection and kinetics in response to tumor Ags remain largely unexplored. In this study, using a novel ex vivo molecular-based approach at the single-cell level, we identified a single, naturally primed T cell clone that dominated the human CD8+ T cell response to the Melan-A/MART-1 Ag. The dominant clone expressed a high-avidity TCR to cognate tumor Ag, efficiently killed tumor cells, and prevailed in the differentiated effector-memory T lymphocyte compartment. TCR sequencing also revealed that this particular clone arose at least 1 year before vaccination, displayed long-term persistence, and efficient homing to metastases. Remarkably, during concomitant vaccination over 3.5 years, the frequency of the pre-existing clone progressively increased, reaching up to 2.5% of the circulating CD8 pool while its effector functions were enhanced. In parallel, the disease stabilized, but subsequently progressed with loss of Melan-A expression by melanoma cells. Collectively, combined ex vivo analysis of T cell differentiation and clonality revealed for the first time a strong expansion of a tumor Ag-specific human T cell clone, comparable to protective virus-specific T cells. The observed successful boosting by peptide vaccination support further development of immunotherapy by including strategies to overcome immune escape.
Regulation of Trafficking Receptor Expression in Human Forkhead Box P3+ Regulatory T Cells
Authors: Lim Hyung W. , Broxmeyer Hal E. , Kim Chang H.
Published: April 2014

Forkhead Box P3+ (FOXP3+) T cells are regulatory cells important for maintaining immune tolerance. While chemokine- and other homing-receptors are important for T cell migration, it has been unclear how they are regulated in FOXP3+ T cells. We thoroughly investigated, ex vivo and in vitro, the regulation of chemokine receptor expression on human FOXP3+ T cells in neonatal cord blood, adult peripheral blood, and tonsils. We found that human FOXP3+ T cells undergo changes in trafficking receptors according to their stages of activation and differentiation. FOXP3+ T cells are divided into CD45RA+ (naive type) and CD45RO+ (memory type) FOXP3+ T cells in neonatal blood, adult blood, and tonsils. CD45RA+FOXP3+ T cells mainly express lymphoid tissue homing receptors (CD62L, CCR7, and CXCR4), while CD45RO+FOXP3+ T cells highly express both Th1 and Th2-associated trafficking receptors along with the lymphoid tissue homing receptors at reduced frequencies. Up-regulation of Th1/Th2-associated trafficking receptors begins with activation of CD45RA+FOXP3+ T cells and is completed after their differentiation to CD45RO+ cells. Some chemokine receptors such as CXCR5 and CXCR6 are preferentially expressed by many FOXP3+ cells at a specific stage (CD69+CD45RO+) in tonsils. Our in vitro differentiation study demonstrated that CD45RA+FOXP3+ T cells indeed undergo chemokine receptor switch from CD45RA+ (secondary lymphoid tissue homing) to CD45RO+ type (lymphoid and nonlymphoid tissue homing). The orderly regulation of trafficking receptors in FOXP3+ T cells according to stages of differentiation and activation is potentially important for their tissue-specific migration and regulation of immune responses in humans.
Characterization of (4-Hydroxy-3-Nitrophenyl)Acetyl (NP)-Specific Germinal Center B Cells and Antigen-Binding B220− Cells after Primary NP Challenge in Mice
Authors: Wolniak Kristy L. , Noelle Randolph J. , Waldschmidt Thomas J.
Published: April 2014

Previous studies examining the primary germinal center (GC) response to SRBC in mice demonstrated a steady ratio of IgM+ to isotype-switched GC B cells and a persistent population of GC B cells with a founder phenotype. These characteristics held true at the inductive, plateau, and dissociative phases of the GC response, suggesting a steady-state environment. To test whether these characteristics apply to the primary response of other T cell-dependent Ags, the present study examined the GC response after challenge with (4-hydroxy-3-nitrophenyl)acetyl (NP) in C57BL/6 mice. Multiparameter flow cytometric analysis was used to assess the phenotype of splenic NP-reactive cells at multiple time points after immunization. Results of these studies demonstrated the characteristics of the SRBC-induced GC reaction to be fully maintained in the NP response. In particular, there was a steady ratio of nonswitched to switched B cells, with the majority of NP-reactive GC B cells displaying IgM. In addition, a substantial frequency of B220− NP-binding cells was observed in the spleen at later time points after NP challenge. Although these cells were IgE+, they were found to express both κ and λ L chains and display the high-affinity IgE Fc (FcεRI) receptor, suggesting that this population is not of B cell origin. Adoptive transfer studies further demonstrated the B220− NP-binding subset to be derived from the myeloid lineage.
TLR7 Ligands Induce Higher IFN-α Production in Females
Authors: Berghöfer Beate , Frommer Ture , Haley Gabriela , Fink Ludger , Bein Gregor , Hackstein Holger
Published: April 2014

IFN-α exercises multiple immune modulatory and antiviral activities and has been suggested to play a critical role in the pathogenesis of systemic lupus erythematosus (SLE). Plasmacytoid dendritic cells (pDCs) release IFN-α upon TLR7 and TLR9 ligation. With respect to the nine times higher incidence of SLE in women and the clinical use of synthetic TLR ligands as novel immune adjuvants, we analyzed IFN-α and TNF-α production in healthy human individuals. Blood samples were incubated with synthetic TLR7 and TLR9 ligands. In three independent groups (n1 = 120, n2 = 101, and n3 = 123), analysis revealed a capacity of female PBLs to produce significantly higher IFN-α levels after TLR7 stimulation (p1 < 0.0000001, p2 < 0.0000001, and p3 < 0.0001) compared with male PBLs. In contrast, no sex differences were evident after TLR9 stimulation. TNF-α production after TLR7 stimulation and also total pDC numbers were not different between females and males. X-inactivation escape of the TLR7 gene was investigated in monoclonal B cell lines and, independently, in pDCs after cell sorting and single-cell picking, indicating regular silencing of one TLR7 allele in females. Additionally, exogenous 17β-estrogen and estrogen receptor antagonism did not indicate a significant role on TLR7-induced IFN-α production. Our data reveal for the first time a profound sex-dependent pathway of TLR7-induced IFN-α with higher production in females. These findings may explain the higher prevalence of SLE in females and the reported decreased therapeutic efficacy of synthetic TLR7 ligands in male individuals.
Screening and Identification of Severe Acute Respiratory Syndrome-Associated Coronavirus-Specific CTL Epitopes
Authors: Zhou Minghai , Xu Dongping , Li Xiaojuan , Li Hongtao , Shan Ming , Tang Jiaren , Wang Min , Wang Fu-Sheng , Zhu Xiaodong , Tao Hua , He Wei , Tien Po , Gao George F.
Published: April 2014

Severe acute respiratory syndrome (SARS) is a highly contagious and life-threatening disease that emerged in China in November 2002. A novel SARS-associated coronavirus was identified as its principal etiologic agent; however, the immunopathogenesis of SARS and the role of special CTLs in virus clearance are still largely uncharacterized. In this study, potential HLA-A*0201-restricted spike (S) and nucleocapsid protein-derived peptides were selected from an online database and screened for potential CTL epitopes by in vitro refolding and T2 cell-stabilization assays. The antigenicity of nine peptides which could refold with HLA-A*0201 molecules was assessed with an IFN-γ ELISPOT assay to determine the capacity to stimulate CTLs from PBMCs of HLA-A2+ SARS-recovered donors. A novel HLA-A*0201-restricted decameric epitope P15 (S411–420, KLPDDFMGCV) derived from the S protein was identified and found to localize within the angiotensin-converting enzyme 2 receptor-binding region of the S1 domain. P15 could significantly enhance the expression of HLA-A*0201 molecules on the T2 cell surface, stimulate IFN-γ-producing CTLs from the PBMCs of former SARS patients, and induce specific CTLs from P15-immunized HLA-A2.1 transgenic mice in vivo. Furthermore, significant P15-specific CTLs were induced from HLA-A2.1-transgenic mice immunized by a DNA vaccine encoding the S protein; suggesting that P15 was a naturally processed epitope. Thus, P15 may be a novel SARS-associated coronavirus-specific CTL epitope and a potential target for characterization of virus control mechanisms and evaluation of candidate SARS vaccines.
Structurally Distinct Ligand-Binding or Ligand-Independent Notch1 Mutants Are Leukemogenic but Affect Thymocyte Development, Apoptosis, and Metastasis Differently
Authors: Priceputu Elena , Bouallaga Isabelle , Zhang YaoPing , Li Xiujie , Chrobak Pavel , Hanna Zaher S. , Poudrier Johanne , Kay Denis G. , Jolicoeur Paul
Published: April 2014

We previously found that provirus insertion in T cell tumors of mouse mammary tumor virus/c-myc transgenic (Tg) mice induced two forms of Notch1 mutations. Type I mutations generated two truncated molecules, one intracellular (IC) (Notch1IC) and one extracellular (Notch1EC), while in type II mutations Notch1 was deleted of its C terminus (Notch1ΔCT). We expressed these mutants in Tg mice using the CD4 promoter. Both Notch1IC and Notch1ΔCT, but not Notch1EC, Tg mice developed double-positive (DP) thymomas. These disseminated more frequently in Notch1ΔCT Tg mice. Double (Notch1IC × myc) or (Notch1ΔCT × myc) Tg mice developed thymoma with a much shorter latency than single Tg mice, providing genetic evidence of a collaboration between these two oncogenes. FACS analysis of preleukemic thymocytes did not reveal major T cell differentiation anomalies, except for a higher number of DP cells and an accumulation of TCRhighCD2highCD25high DP cells in Notch1IC, and less so in Notch1ΔCT Tg mice. This was associated with enhanced in vivo thymocyte proliferation. However, Notch1IC, but not Notch1ΔCT, DP thymocytes were protected against apoptosis induced in vivo by dexamethasone and anti-CD3 and in vitro by anti-CD3/CD28 Abs. This indicates that the C terminus of Notch1 and/or the conserved regulation by its ligands have a significant impact on the induced T cell phenotype. Therefore, Notch1IC and Notch1ΔCT behave as oncogenes for T cells. Because these two Notch1 mutations are very similar to those described in some forms of human T cell leukemia, these Tg mice may represent relevant models of these human leukemias.