bluuebunny/crossref_metadata_2025 huggingface dataset:
The Transcription Factor Nrf2 Is a Therapeutic Target against Brain Inflammation
Authors: Innamorato Nadia G. , Rojo Ana I. , García-Yagüe Ángel J. , Yamamoto Masayuki , de Ceballos María L. , Cuadrado Antonio
Published: April 2014

Because chronic neuroinflammation is a hallmark of neurodegenerative diseases and compromises neuron viability, it is imperative to discover pharmacologic targets to modulate the activation of immune brain cells, the microglia. In this study, we identify the transcription factor Nrf2, guardian of redox homeostasis, as such target in a model of LPS-induced inflammation in mouse hippocampus. Nrf2 knockout mice were hypersensitive to the neuroinflammation induced by LPS, as determined by an increase in F4/80 mRNA and protein, indicative of an increase in microglial cells, and in the inflammation markers inducible NO synthase, IL-6, and TNF-α, compared with the hippocampi of wild-type littermates. The aliphatic isothiocyanate sulforaphane elicited an Nrf2-mediated antioxidant response in the BV2 microglial cell line, determined by flow cytometry of cells incubated with the redox sensitive probe dihydrodichlorofluorescein diacetate, and by the Nrf2-dependent induction of the phase II antioxidant enzyme heme oxygenase-1. Animals treated with sulforaphane displayed a 2–3-fold increase in heme oxygenase-1, a reduced abundance of microglial cells in the hippocampus and an attenuated production of inflammation markers (inducible NO synthase, IL-6, and TNF-α) in response to LPS. Considering that release of reactive oxygen species is a property of activated microglia, we propose a model in which late induction of Nrf2 intervenes in the down-regulation of microglia. This study opens the possibility of targeting Nrf2 in brain as a means to modulate neuroinflammation.
Transcriptome Analysis Reveals Human Cytomegalovirus Reprograms Monocyte Differentiation toward an M1 Macrophage
Authors: Chan Gary , Bivins-Smith Elizabeth R. , Smith M. Shane , Smith Patrick M. , Yurochko Andrew D.
Published: April 2014

Monocytes are primary targets for human CMV (HCMV) infection and are proposed to be responsible for hematogenous dissemination of the virus. Monocytes acquire different functional traits during polarization to the classical proinflammatory M1 macrophage or the alternative antiinflammatory M2 macrophage. We hypothesized that HCMV induced a proinflammatory M1 macrophage following infection to promote viral dissemination because, biologically, a proinflammatory state provides the tools to drive infected monocytes from the blood into the tissue. To test this hypothesis of monocyte conversion from a normal quiescent phenotype to an inflammatory phenotype, we used Affymetrix Microarray to acquire a transcriptional profile of infected monocytes at a time point our data emphasized is a key temporal regulatory point following infection. We found that HCMV significantly up-regulated 583 (5.2%) of the total genes and down-regulated 621 (5.5%) of the total genes ≥1.5-fold at 4 h postinfection. Further ontology analysis revealed that genes implicated in classical M1 macrophage activation were stimulated by HCMV infection. We found that 65% of genes strictly associated with M1 polarization were up-regulated, while only 4% of genes solely associated with M2 polarization were up-regulated. Analysis of the monocyte chemokinome at the transcriptional level showed that 44% of M1 and 33% of M2 macrophage chemokines were up-regulated. Proteomic analysis using chemokine Ab arrays confirmed the secretion of these chemotactic proteins from HCMV-infected monocytes. Overall, the results identify that the HCMV-infected monocyte transcriptome displayed a unique M1/M2 polarization signature that was skewed toward the classical M1 activation phenotype.
Fetal Exposure to High-Avidity TCR Ligand Enhances Expansion of Peripheral T Regulatory Cells
Authors: Yu Ping , Haymaker Cara L. , Divekar Rohit D. , Ellis Jason S. , Hardaway John , Jain Renu , Tartar Danielle M. , Hoeman Christine M. , Cascio Jason A. , Ostermeier Austin , Zaghouani Habib
Published: April 2014

Lately, it has become clear that regulatory T cells (Tregs) play a major role in the maintenance of peripheral tolerance and control of autoimmunity. Despite these critical functions, the process underlying the development of Tregs remains largely undefined. Herein, altered peptide ligand (APL) variants derived from the proteolipid protein-1 (PLP1) epitope were expressed on immunoglobulins (Igs) and the resulting Ig-APLs were used to deliver the APLs from mother to fetus through the maternal placenta to influence thymic T cell selection. This delivery system was then adapted to the SJL/J mouse, a strain that expresses only the DM20 form of PLP, which lacks the dominant PLP1 epitope in the thymus during fetal and neonatal development. This model, which restores thymic T cell selection for PLP1, was then used to determine whether affinity plays a role in the development of Tregs. The findings show that fetal exposure to low-affinity peptide ligand was unable to drive development of Tregs while variants with higher affinity to the TCR resulted in significant seeding of the periphery with mature, naive Tregs. Thus, contrary to pathogenic T cells, Tregs require avid TCR-ligand interaction to undergo thymic development and maturation.
The Anergic State in Sarcoidosis Is Associated with Diminished Dendritic Cell Function
Authors: Mathew Sneha , Bauer Kristy L. , Fischoeder Arne , Bhardwaj Nina , Oliver Stephen J.
Published: April 2014

Sarcoidosis is a chronic inflammatory disease of unknown cause, characterized by granuloma formation similar to tuberculosis, but without clear evidence of a microbial infection. Because sarcoidosis is linked with clinical anergy and other evidence of diminished cellular immunity, we hypothesized that decreased skin delayed-type hypersensitivity (DTH) responses to recall Ags in affected individuals would be associated with decreased function of their blood dendritic cells (DCs). Our study involved ex vivo isolation, phenotyping, and functional testing of myeloid DCs (mDCs), plasmacytoid DCs, and T lymphocytes from blood of normal healthy volunteers and sarcoidosis subjects with active, untreated pulmonary disease. We found mDC function in the allogeneic MLR directly corresponded to the magnitude of skin DTH reactions to recall Ags in both sarcoidosis subjects and normal volunteers. However, both of these outcomes were significantly decreased in the sarcoidosis group. Diminished mDC function occurred despite up-regulated costimulatory and maturation markers. Clinical relevance is suggested by the inverse relationship between both mDC allogeneic responses and skin DTH responses with clinical disease severity as measured by chest radiograms. Because granulomas form when cellular immunity fails to clear antigenic stimuli, attenuated mDC function in sarcoidosis may contribute to susceptibility and persistence of the chronic inflammation characteristic of this disease.
Cutting Edge: Experimentally Induced Immune Activation in Natural Hosts of Simian Immunodeficiency Virus Induces Significant Increases in Viral Replication and CD4+ T Cell Depletion
Authors: Pandrea Ivona , Gaufin Thaidra , Brenchley Jason M. , Gautam Rajeev , Monjure Christopher , Gautam Aarti , Coleman Clint , Lackner Andrew A. , Ribeiro Ruy M. , Douek Daniel C. , Apetrei Cristian
Published: April 2014

Chronically SIVagm-infected African green monkeys (AGMs) have a remarkably stable nonpathogenic disease course, with levels of immune activation in chronic SIVagm infection similar to those observed in uninfected monkeys and with stable viral loads for long periods of time. In vivo administration of LPS or an IL-2/diphtheria toxin fusion protein (Ontak) to chronically SIVagm-infected AGMs triggered increases in immune activation and subsequently of viral replication and depletion of intestinal CD4+ T cells. Our study indicates that circulating microbial products can increase viral replication by inducing immune activation and increasing the number of viral target cells, thus demonstrating that immune activation and T cell proliferation are key factors in AIDS pathogenesis.
Cutting Edge: Eosinophils Do Not Contribute to Respiratory Syncytial Virus Vaccine-Enhanced Disease
Authors: Castilow Elaine M. , Legge Kevin L. , Varga Steven M.
Published: April 2014

Respiratory syncytial virus (RSV) infection of BALB/c mice previously immunized with a recombinant vaccinia virus (vacv) expressing the attachment (G) protein of RSV (vacvG) results in pulmonary eosinophilia, which mimics the response of formalin-inactivated RSV-vaccinated children, as well as increased weight loss, clinical illness, and enhanced pause (Penh). We show that RSV infection of eosinophil-deficient mice previously immunized with vacvG results in the development of increased weight loss, clinical illness, and Penh similar to that in wild-type controls. These measures of RSV vaccine-enhanced disease are dependent upon STAT4. Interestingly, neither IL-12 nor IL-23, the two most common STAT4-activating cytokines, proved necessary for the development of disease. We demonstrate that IFN-γ, which is produced following STAT4 activation, contributes to clinical illness and increased Penh, but not weight loss. Our results have important implications for future RSV vaccine design, suggesting that enhancing a Th1 response may exacerbate disease.
Cutting Edge: FimH Adhesin of Type 1 Fimbriae Is a Novel TLR4 Ligand
Authors: Mossman Karen L. , Mian M. Firoz , Lauzon Nicole M. , Gyles Carlton L. , Lichty Brian , Mackenzie Randy , Gill Navkiran , Ashkar Ali A.
Published: April 2014

Several TLR ligands of bacterial origin induce innate immune responses. Although FimH, the adhesin portion of type 1 fimbria, plays an important role in the pathogenicity of some Gram-negative bacteria, its ability to stimulate the innate immune system via TLR signaling remains unclear. In this study we report that FimH induces potent innate responses in a MyD88-dependent fashion. The FimH-induced innate activity was restricted to cells expressing TLR4. In addition, FimH was able to bind directly to TLR4. More importantly, cells unresponsive to LPS were responsive to FimH and the presence or absence of MD-2 and CD14 had no effect on FimH activity. Our data suggest that TLR4 is a functional receptor for the adhesin portion of bacterial type 1 fimbria.
Cutting Edge: Programmed Death-1/Programmed Death Ligand 1 Interaction Regulates the Induction and Maintenance of Invariant NKT Cell Anergy
Authors: Chang Woo-Sung , Kim Ji-Yeon , Kim Yeon-Jeong , Kim Yun-Sun , Lee Jung-Mi , Azuma Miyuki , Yagita Hideo , Kang Chang-Yuil
Published: April 2014

Invariant NKT (iNKT) cells are a distinct subset of T lymphocytes that recognize glycolipid Ags. Upon TCR stimulation, iNKT cells promptly secrete a wide range of cytokines and therefore have been investigated as a target for immunotherapy. However, after primary activation, iNKT cells become hyporesponsive toward their ligand (anergy). The further mechanism behind iNKT cell anergy is poorly understood. We found that a low level of programmed death-1 (PD-1) was constitutively expressed on iNKT cells and that PD-1 expression was increased after stimulation and lasted at least 2 mo. Moreover, not only did blocking of the PD-1/PD ligand 1 (PD-L1) pathway prevent the induction of anergy in iNKT cells, but anergic iNKT cells also recovered responsiveness and these “rescued” cells efficiently mediated antitumor immunity. Our findings suggest that the PD-1/PD-L1 interaction is essential for the induction and maintenance of iNKT cell anergy.
Reversible Blockade of Thymic Output: An Inherent Part of TLR Ligand-Mediated Immune Response
Authors: Démoulins Thomas , Abdallah Ali , Kettaf Nadia , Baron Marie-Laurence , Gerarduzzi Casimiro , Gauchat Dominique , Gratton Sophie , Sékaly Rafick-Pierre
Published: April 2014

TLRs constitute a first set of sensors that detect viral nucleic acids including dsRNA which triggers TLR3. We report the early, direct, and detrimental effect of polyinosine-polycytidilic acid treatment on T cell development. Inhibition of thymopoiesis was targeted to several thymocyte subpopulations. First, both a blockade of the double negative (DN)1-DN2 transition and a severe down-regulation of DN3-DN4 thymocyte proliferation were observed. In addition, an important decrease in the absolute numbers of double-positive thymocytes, concomitant with an increase in frequencies of apoptotic cells in this population were shown. This inhibition of thymopoiesis resulted in a reduced thymic output, as evidenced by a drop of the absolute numbers of naive T cells and TCR excision circles levels. The decrease in thymic cellularity and defects in thymic development were severely reduced, but not completely abolished in IFN-α/βR−/− mice, showing a direct contribution of type I IFNs, known to be massively up-regulated in viral infections, to the inhibition of T cell development. Strikingly, the TCR repertoire in treated mice was biased toward shorter CDR3 lengths as a result of a decreased expression of TdT and Rag2. However, thymic integrity remained intact since thymopoiesis was restored both quantitatively and qualitatively 14 days after the cessation of polyinosine-polycytidilic acid treatment. These results demonstrate a novel immunomodulatory role for virally encoded TLR ligands and RNA sensors; they further illustrate the diversity of mechanisms that viruses use to interfere with the development of a pathogen-specific immune responses.
Adaptable TCR Avidity Thresholds for Negative Selection
Authors: Stojakovic Milica , Salazar-Fontana Laura I. , Tatari-Calderone Zohreh , Badovinac Vladimir P. , Santori Fabio R. , Kovalovsky Damian , Sant'Angelo Derek , Harty John T. , Vukmanovic Stanislav
Published: April 2014

Central tolerance plays a significant role in preventing autoimmune diseases by eliminating T cells with high and intermediate avidity for self. To determine the manner of setting the threshold for deletion, we created a unique transgenic mouse strain with a diverse T cell population and globally increased TCR avidity for self-peptide/MHC complexes. Despite the adaptations aimed at reducing T cell reactivity (reduced TCR levels and increased levels of TCR signaling inhibitor CD5), transgenic mice displayed more severe experimental allergic encephalomyelitis and lupus. The numbers and activity of natural (CD4+CD25+) regulatory T cells were not altered. These findings demonstrate that the threshold for deletion is adaptable, allowing survival of T cells with higher avidity when TCR avidity is globally increased.
The Host Environment Regulates the Function of CD8+ Graft-versus-Host-Reactive Effector Cells
Authors: Chakraverty Ronjon , Flutter Barry , Fallah-Arani Farnaz , Eom Hyeon-Seok , Means Terry , Andreola Giovanna , Schwarte Sebastian , Buchli Jennifer , Cotter Pete , Zhao Guiling , Sykes Megan
Published: April 2014

We have examined how the host environment influences the graft-vs-leukemia (GVL) response following transfer of donor T cells to allogeneic chimeras. Donor T cells induce significant GVL when administered in large numbers to established mixed chimeras (MC). However, when using limiting numbers of T cells, we found that late transfer to MC induced less GVL than did early transfer to freshly irradiated allogeneic recipients. Late donor T cell transfer to MC was associated with marked accumulation of anti-host CD8 cells within the spleen, but delayed kinetics of differentiation, reduced expression of effector molecules including IFN-γ, impaired cytotoxicity, and higher rates of sustained apoptosis. Furthermore, in contrast to the spleen, we observed a significant delay in donor CD8 cell recruitment to the bone marrow, a key location for hematopoietic tumors. Increasing the numbers of T cells transferred to MC led to the enhancement of CTL activity and detectable increases in absolute numbers of IFN-γ+ cells without inducing graft-vs-host disease (GVHD). TLR-induced systemic inflammation accelerated differentiation of functional CTL in MC but was associated with severe GVHD. In the absence of inflammation, both recipient T and non-T cell populations impeded the full development of GVHD-inducing effector function. We conclude that per-cell deficits in the function of donor CD8 cells activated in MC may be overcome by transferring larger numbers of T cells without inducing GVHD.
MHC Variant Peptide-Mediated Anergy of Encephalitogenic T Cells Requires SHP-1
Authors: Wasserman Heather A. , Beal Carrie D. , Zhang Yan , Jiang Ning , Zhu Cheng , Evavold Brian D.
Published: April 2014

Our lab has demonstrated that encephalitogenic T cells can be effectively anergized by treatment with MHC variant peptides, which are analogues of immunogenic peptides containing an amino acid substitution at an MHC anchor residue. The MHC variant peptide of myelin oligodendrocyte glycoprotein (MOG)35–55 proves an effective treatment as it does not induce symptoms of experimental autoimmune encephalomyelitis and fails to recruit macrophages or MOG35–55-specific T cells to the CNS. In this study, we sought to characterize the signaling pathways required for the induction of anergy by building upon the observations identifying the tyrosine phosphatase SHP-1 as a critical regulator of T cell responsiveness. Motheaten viable heterozygous mice, which contain a mutation in the SHP-1 gene resulting in a reduction in functional SHP-1, were challenged with MOG35–55 or the MOG35–55 MHC variant 45D. These mice display symptoms of experimental autoimmune encephalomyelitis upon immunization with MHC variant peptide and have significant CNS infiltration of tetramer-positive CD4+ cells and macrophages, unlike B6 mice challenged with the variant peptide. The effects of SHP-1 are directly on the T cell as Motheaten viable heterozygous mice autoreactive T cells are not anergized in vitro. Lastly, we demonstrate no distinguishable difference in the initial interaction between the TCR and agonist or MHC variant. Rather, an unstable interaction between peptide and MHC attenuates the T cell response, seen in a decreased half-life relative to MOG35–55. These results identify SHP-1 as a mediator of T cell anergy induced by destabilized peptide:MHC complexes.
Polysialic Acid, a Glycan with Highly Restricted Expression, Is Found on Human and Murine Leukocytes and Modulates Immune Responses
Authors: Drake Penelope M. , Nathan Jay K. , Stock Christina M. , Chang Pamela V. , Muench Marcus O. , Nakata Daisuke , Reader J. Rachel , Gip Phung , Golden Kevin P. K. , Weinhold Birgit , Gerardy-Schahn Rita , Troy Frederic A. , Bertozzi Carolyn R.
Published: April 2014

Polysialic acid (polySia) is a large glycan with restricted expression, typically found attached to the protein scaffold neural cell adhesion molecule (NCAM). PolySia is best known for its proposed role in modulating neuronal development. Its presence and potential functions outside the nervous systems are essentially unexplored. Herein we show the expression of polySia on hematopoietic progenitor cells, and demonstrate a role for this glycan in immune response using both acute inflammatory and tumor models. Specifically, we found that human NK cells modulate expression of NCAM and the degree of polymerization of its polySia glycans according to activation state. This contrasts with the mouse, where polySia and NCAM expression are restricted to multipotent hematopoietic progenitors and cells developing along a myeloid lineage. Sialyltransferase 8Sia IV−/− mice, which lacked polySia expression in the immune compartment, demonstrated an increased contact hypersensitivity response and decreased control of tumor growth as compared with wild-type animals. This is the first demonstration of polySia expression and regulation on myeloid cells, and the results in animal models suggest a role for polySia in immune regulation.
Antiviral-Activated Dendritic Cells: A Paracrine-Induced Response State
Authors: Bordería Antonio V. , Hartmann Boris M. , Fernandez-Sesma Ana , Moran Thomas M. , Sealfon Stuart C.
Published: April 2014

Infection of immature dendritic cells (DCs) by virus stimulates their maturation into APC. Infected DCs can also expose uninfected DCs to a panoply of cytokines/chemokines via paracrine signaling. Mathematical modeling suggests that a high rate of paracrine signaling is likely to occur among DCs located in three-dimensional space. Relatively little is known about how secreted factors modify the early response to virus infection. We used a transwell experimental system that allows passage of secreted factors, but not direct contact, between virus-infected DCs and uninfected DCs to investigate paracrine signaling responses. Paracrine signaling from infected DCs induced an antiviral-primed DC state distinct from that of mature virus-infected DCs that we refer to as antiviral-activated DCs (AVDCs). AVDCs had increased surface MHC class II and CD86 levels, but in contrast to virus-infected DCs, their MHC class I levels were unchanged. Imaging flow cytometry showed that AVDCs had an increased rate of phagocytosis compared with naive DCs. Experiments with IFN-β cytokine indicated that it may be responsible for CD86, but not MHC class II regulation in AVDCs. Both IFN-inducible and IFN-independent genes are up-regulated in AVDCs. Notably, AVDCs are relatively resistant to virus infection in comparison to naive DCs and achieve accelerated and augmented levels of costimulatory molecule expression with virus infection. AVDCs show a distinct antiviral-primed state of DC maturation mediated by DC paracrine signaling. Although further in vivo study is needed, the characteristics of the AVDC suggest that it is well suited to play a role in the early innate-adaptive transition of the immune system.
PI4P5-Kinase Iα Is Required for Efficient HIV-1 Entry and Infection of T Cells
Authors: Barrero-Villar Marta , Barroso-González Jonathan , Cabrero J. R. , Gordón-Alonso Mónica , Álvarez-Losada Susana , Muñoz-Fernández M. A. , Sánchez-Madrid Francisco , Valenzuela-Fernández Agustín
Published: April 2014

HIV-1 envelope (Env) triggers membrane fusion between the virus and the target cell. The cellular mechanism underlying this process is not well known. Phosphatidylinositol 4,5-bisphosphate (PIP2) is known to be important for the late steps of the HIV-1 infection cycle by promoting Gag localization to the plasma membrane during viral assembly, but it has not been implicated in early stages of HIV-1 membrane-related events. In this study, we show that binding of the initial HIV-1 Env-gp120 protein induces PIP2 production in permissive lymphocytes through the activation of phosphatidylinositol-4-phosphate 5-kinase (PI4P5-K) Iα. Overexpression of wild-type PI4P5-K Iα increased HIV-1 Env-mediated PIP2 production and enhanced viral replication in primary lymphocytes and CEM T cells, whereas PIP2 production and HIV-1 infection were both severely reduced in cells overexpressing the kinase-dead mutant D227A (D/A)-PI4P5-K Iα. Similar results were obtained with replicative and single-cycle HIV-1 particles. HIV-1 infection was also inhibited by knockdown of endogenous expression of PI4P5-K Iα. These data indicate that PI4P5-K Iα-mediated PIP2 production is crucial for HIV-1 entry and the early steps of infection in permissive lymphocytes.
Transgenic Mice Expressing Dominant-Negative Bright Exhibit Defects in B1 B Cells
Authors: Nixon Jamee C. , Ferrell Scott , Miner Cathrine , Oldham Athenia L. , Hochgeschwender Ute , Webb Carol F.
Published: April 2014

The transcription factor Bright up-regulates Ig H chain production from select V region promoters and requires Bright dimerization, Bruton’s tyrosine kinase (Btk), and the Btk substrate, TFII-I, for this activity. Defects in Btk cause X-linked immunodeficiency disease in mice and humans. Btk-deficient mice exhibit decreased serum IgM production, B cell developmental blocks, absence of peritoneal B1 cells, and subnormal immune responses against Ags, including phosphorylcholine, which confer protection against Streptococcus pneumoniae. Transgenic mice expressing dominant-negative Bright share similarities with Btk-deficient mice, including decreased serum IgM, poor anti-phosphorylcholine responses, and slightly reduced numbers of mature B cells. Although dominant-negative Bright mice developed B1 B cells, these were functionally deficient in Ig secretion. These data suggest a mechanistic explanation for the abnormal responses to phosphorylcholine observed in Btk-deficient mice, and indicate that Bright functions in a subset of Btk-dependent pathways in vivo, particularly those responses dominated by B1 B cells.
CD8+CD205+ Splenic Dendritic Cells Are Specialized to Induce Foxp3+ Regulatory T Cells
Authors: Yamazaki Sayuri , Dudziak Diana , Heidkamp Gordon F. , Fiorese Christopher , Bonito Anthony J. , Inaba Kayo , Nussenzweig Michel C. , Steinman Ralph M.
Published: April 2014

Foxp3+CD25+CD4+ regulatory T cells (Treg) mediate immunological self-tolerance and suppress immune responses. A subset of dendritic cells (DCs) in the intestine is specialized to induce Treg in a TGF-β- and retinoic acid-dependent manner to allow for oral tolerance. In this study we compare two major DC subsets from mouse spleen. We find that CD8+ DEC-205/CD205+ DCs, but not the major fraction of CD8− DC inhibitory receptor-2 (DCIR2)+ DCs, induce functional Foxp3+ Treg from Foxp3− precursors in the presence of low doses of Ag but without added TGF-β. CD8+CD205+ DCs preferentially express TGF-β, and the induction of Treg by these DCs in vitro is blocked by neutralizing Ab to TGF-β. In contrast, CD8−DCIR2+ DCs better induce Foxp3+ Treg when exogenous TGF-β is supplied. In vivo, CD8+CD205+ DCs likewise preferentially induce Treg from adoptively transferred, Ag-specific DO11.10 RAG−/− Foxp3−CD4+ T cells, whereas the CD8−DCIR2+ DCs better stimulate natural Foxp3+ Treg. These results indicate that a subset of DCs in spleen, a systemic lymphoid organ, is specialized to differentiate peripheral Foxp3+ Treg, in part through the endogenous formation of TGF-β. Targeting of Ag to these DCs might be useful for inducing Ag-specific Foxp3+ Treg for treatment of autoimmune diseases, transplant rejection, and allergy.
Rapid and Selective Expansion of Nonclonotypic T Cells in Regulatory T Cell-Deficient, Foreign Antigen-Specific TCR-Transgenic Scurfy Mice: Antigen-Dependent Expansion and TCR Analysis
Authors: Sharma Rahul , Ju Angela Chiao-Ying , Kung John T. , Fu Shu Man , Ju Shyr-Te
Published: April 2014

Foreign Ag-specific TCR-transgenic (Tg) mice contain a small fraction of T cells bearing the endogenous Vβ and Vα chains as well as a population expressing an intermediate level of Tg TCR. Importantly, these minor nonclonotypic populations contain ≥99% of the CD4+Foxp3+ regulatory T cells (Treg) and, despite low overall Treg expression, peripheral tolerance is maintained. In the OT-II TCR (OVA-specific, Vβ5highVα2high) Tg scurfy (Sf) mice (OT-II Sf) that lack Treg, nonclonotypic T cells markedly expanded in the periphery but not in the thymus. Expanded T cells expressed memory/effector phenotype and were enriched in blood and inflamed lungs. In contrast, Vβ5highVα2high clonotypic T cells were not expanded, displayed the naive phenotype, and found mainly in the lymph nodes. Importantly, Vβ5neg T cells were able to transfer multiorgan inflammation in Rag1−/− recipients. T cells bearing dual TCR (dual Vβ or dual Vα) were demonstrated frequently in the Vβ5int and Vα2int populations. Our study demonstrated that in the absence of Treg, the lack of peripheral expansion of clonotypic T cells is due to the absence of its high-affinity Ag OVA. Thus, the rapid expansion of nonclonotypic T cells in OT-II Sf mice must require Ag (self and foreign) with sufficient affinity. Our study has implications with respect to the roles of Ag and dual TCR in the selection and regulation of Treg and Treg-controlled Ag-dependent T cell expansion in TCR Tg and TCR Tg Sf mice, respectively.
Imatinib Mesylate Inhibits CD4+CD25+ Regulatory T Cell Activity and Enhances Active Immunotherapy against BCR-ABL− Tumors
Authors: Larmonier Nicolas , Janikashvili Nona , LaCasse Collin James , Larmonier Claire Billerey , Cantrell Jessica , Situ Elaine , Lundeen Tamara , Bonnotte Bernard , Katsanis Emmanuel
Published: April 2014

Imatinib mesylate (Gleevec, STI571), a selective inhibitor of a restricted number of tyrosine kinases, has been effectively used for the treatment of Philadelphia chromosome-positive leukemias and gastrointestinal stromal tumors. Imatinib may also directly influence immune cells. Suppressive as well as stimulating effects of this drug on CD4+ and CD8+ T lymphocytes or dendritic cells have been reported. In the current study, we have investigated the influence of imatinib mesylate on CD4+CD25+FoxP3+ regulatory T cells (Treg), a critical population of lymphocytes that contributes to peripheral tolerance. Used at concentrations achieved clinically, imatinib impaired Treg immunosuppressive function and FoxP3 expression but not production of IL-10 and TGF-β in vitro. Imatinib significantly reduced the activation of the transcription factors STAT3 and STAT5 in Treg. Analysis of Treg TCR-induced signaling cascade indicated that imatinib inhibited phosphorylation of ZAP70 and LAT. Substantiating these observations, imatinib treatment of mice decreased Treg frequency and impaired their immunosuppressive function in vivo. Furthermore, imatinib mesylate significantly enhanced antitumor immune responses to dendritic cell-based immunization against an imatinib-resistant BCR-ABL negative lymphoma. The clinical applications of imatinib mesylate might thus be expanded with its use as a potent immunomodulatory agent targeting Treg in cancer immunotherapy.
Targeting CD4 Coreceptor Expression to Postselection Thymocytes Reveals That CD4/CD8 Lineage Choice Is neither Error-Prone nor Stochastic
Authors: Adoro Stanley , Erman Batu , Sarafova Sophia D. , Van Laethem François , Park Jung-Hyun , Feigenbaum Lionel , Singer Alfred
Published: April 2014

The mechanism by which CD4/CD8 lineage choice is coordinated with TCR specificity during positive selection remains an unresolved problem in immunology. The stochastic/selection model proposes that CD4/CD8 lineage choice in TCR-signaled CD4+CD8+ thymocytes occurs randomly and therefore is highly error-prone. This perspective is strongly supported by “coreceptor rescue” experiments in which transgenic CD4 coreceptors were ectopically expressed on thymocytes throughout their development and caused significant numbers of cells bearing MHC-II-specific TCR to differentiate into mature, CD8 lineage T cells. However, it is not known if forced coreceptor expression actually rescued positively selected thymocytes making an incorrect lineage choice or if it influenced developing thymocytes into making an incorrect lineage choice. We have now reassessed coreceptor rescue and the concept that lineage choice is highly error-prone with a novel CD4 transgene (referred to as E8I-CD4) that targets expression of transgenic CD4 coreceptors specifically to thymocytes that have already undergone positive selection and adopted a CD8 lineage fate. Unlike previous CD4 transgenes, the E8I-CD4 transgene has no effect on early thymocyte development and cannot itself influence CD4/CD8 lineage choice. We report that the E8I-CD4 transgene did in fact induce expression of functional CD4 coreceptor proteins on newly arising CD8 lineage thymocytes precisely at the point in thymic development that transgenic CD4 coreceptors would putatively rescue MHC-II-specific thymocytes that incorrectly adopted the CD8 lineage. However, the E8I-CD4 transgene did not reveal any MHC-II-selected thymocytes that adopted the CD8 lineage fate. These results demonstrate that CD4/CD8 lineage choice is neither error-prone nor stochastic.
Tetraspanins Regulate ADAM10-Mediated Cleavage of TNF-α and Epidermal Growth Factor
Authors: Arduise Cécile , Abache Toufik , Li Lei , Billard Martine , Chabanon Aurélie , Ludwig Andreas , Mauduit Philippe , Boucheix Claude , Rubinstein Eric , Le Naour François
Published: April 2014

Several cytokines and growth factors are released by proteolytic cleavage of a membrane-anchored precursor, through the action of ADAM (a disintegrin and metalloprotease) metalloproteases. The activity of these proteases is regulated through largely unknown mechanisms. In this study we show that Ab engagement of several tetraspanins (CD9, CD81, CD82) increases epidermal growth factor and/or TNF-α secretion through a mechanism dependent on ADAM10. The effect of anti-tetraspanin mAb on TNF-α release is rapid, not relayed by intercellular signaling, and depends on an intact MEK/Erk1/2 pathway. It is also associated with a concentration of ADAM10 in tetraspanin-containing patches. We also show that a large fraction of ADAM10 associates with several tetraspanins, indicating that ADAM10 is a component of the “tetraspanin web.” These data show that tetraspanins regulate the activity of ADAM10 toward several substrates, and illustrate how membrane compartmentalization by tetraspanins can control the function of cell surface proteins such as ectoproteases.
CD45 Down-Regulates Lck-Mediated CD44 Signaling and Modulates Actin Rearrangement in T Cells
Authors: Wong Nelson K. Y. , Lai Jacqueline C. Y. , Birkenhead Darlene , Shaw Andrey S. , Johnson Pauline
Published: April 2014

The tyrosine phosphatase CD45 dephosphorylates the negative regulatory tyrosine of the Src family kinase Lck and plays a positive role in TCR signaling. In this study we demonstrate a negative regulatory role for CD45 in CD44 signaling leading to actin rearrangement and cell spreading in activated thymocytes and T cells. In BW5147 T cells, CD44 ligation led to CD44 and Lck clustering, which generated a reduced tyrosine phosphorylation signal in CD45+ T cells and a more sustained, robust tyrosine phosphorylation signal in CD45− T cells. This signal resulted in F-actin ring formation and round spreading in the CD45+ cells and polarized, elongated cell spreading in CD45− cells. The enhanced signal in the CD45− cells was consistent with enhanced Lck Y394 phosphorylation compared with the CD45+ cells where CD45 was recruited to the CD44 clusters. This enhanced Src family kinase-dependent activity in the CD45− cells led to PI3K and phospholipase C activation, both of which were required for elongated cell spreading. We conclude that CD45 induces the dephosphorylation of Lck at Y394, thereby preventing sustained Lck activation and propose that the amplitude of the Src family kinase-dependent signal regulates the outcome of CD44-mediated signaling to the actin cytoskeleton and T cell spreading.
Negative Regulation of TCR Signaling by Linker for Activation of X Cells via Phosphotyrosine-Dependent and -Independent Mechanisms
Authors: Shapiro Michael J. , Nguyen Chau T. , Aghajanian Haig , Zhang Weiguo , Shapiro Virginia Smith
Published: April 2014

The activation of T cells and the initiation of an immune response is tightly controlled through the crosstalk of both positive and negative regulators. Two adaptors that function as negative regulators of T cell activation are adaptor in lymphocytes of unknown function X (ALX) and linker for activation of X cell (LAX). Previously, we showed that T cells from mice deficient in ALX and LAX display similar hyperresponsiveness, with increased IL-2 production and proliferation upon TCR/CD28 stimulation, and that these adaptors physically associate. In this study, we analyze the nature of the association between ALX and LAX. We demonstrate that this association occurs in the absence of TCR/CD28 signaling via a mechanism independent of both tyrosine phosphorylation of LAX and the SH2 domain of ALX. Cotransfection of ALX with LAX resulted in LAX tyrosine phosphorylation in the absence of TCR/CD28 stimulation. ALX-mediated LAX phosphorylation depends upon the ALX SH2 domain, which functions to recruit Lck to LAX. We also show that LAX, like ALX, can inhibit RE/AP reporter activation. However, in contrast to its inhibition of NFAT, the inhibition of RE/AP by LAX is independent of its tyrosine phosphorylation. Therefore, it can be concluded that inhibition of signaling events involved in T cell activation by LAX occurs through mechanisms both dependent on and independent of its tyrosine phosphorylation.
Processing of an Antigenic Sequence from IgG Constant Domains for Presentation by MHC Class II
Authors: Flobakk Morten , Rasmussen Ingunn B. , Lunde Elin , Frigstad Terje , Berntzen Gøril , Michaelsen Terje E. , Bogen Bjarne , Sandlie Inger
Published: April 2014

Targeting of T cell epitopes to APC enhances T cell responses. We used an APC-specific Ab (anti-IgD) and substituted either of 18 loops connecting β strands in human IgG constant H (CH) domains with a characterized T cell peptide epitope. All Ab-epitope fusion molecules were secreted from producing cells except IgG-loop 2(BC)CH1, and comparing levels, a hierarchy appeared with fusions involving CH2≥CH1>CH3. Within each domain, fusion at loop 6(FG) showed best secretion, while low secretion correlated with the substitution of native loops that contain conserved amino acids buried within the folded molecule. Comparing the APC-specific rAb molecules for their ability to induce T cell activation in vitro, the six mutants with epitope in CH2 were the most effective, with loop 4CH2 ranking on top. The CH1 mutants were more resistant to processing, and the loop 6CH1 mutant only induced detectable activation. The efficiency of the CH3 mutants varied, with loop 6CH3 being the least effective and equal to loop 6 CH1. Considering both rAb secretion level and T cell activation efficiency, a total of eight loops may carry T cell epitopes to APC for processing and presentation to T cells, namely, all in CH2 in addition to loop 6 in CH1 and CH3. Comparing loop 4CH2 with loop 6CH1 mutants after injection of Ab in BALB/c mice, the former was by far the most efficient and induced specific T cell activation at concentrations at least 100-fold lower than loop 6CH1.
NKG2D-RAE-1 Receptor-Ligand Variation Does Not Account for the NK Cell Defect in Nonobese Diabetic Mice
Authors: Maier Lisa M. , Howlett Sarah K. , Rainbow Kara M. , Clark Jan , Howson Joanna M. M. , Todd John A. , Wicker Linda S.
Published: April 2014

NK cells from NOD mice induced with poly(I:C) in vivo exhibit low cytotoxicity against a range of target cells, but the genetic mechanisms controlling this defect are yet to be elucidated. Defects in the expression of NKG2D and its ligands, the RAE-1 molecules, have been hypothesized to contribute to the reduced NK function present in NOD mice. In this study, we show that segregation of the NK-mediated killing phenotype did not correlate with the NOD Raet1 haplotype and that the large alterations in NKG2D expression previously reported on NK cells expanded in vitro were not observed in primary, poly(I:C)-elicited NK cells in vivo. Additional studies indicate a complex genetic control of defective NOD NK cells including genes linked to the MHC and possibly those that are associated with an altered cytokine response to the TLR3-agonist poly(I:C).
Induction of TRAIL- and TNF-α-Dependent Apoptosis in Human Monocyte-Derived Dendritic Cells by Microfilariae of Brugia malayi
Authors: Semnani Roshanak Tolouei , Venugopal Priyanka Goel , Mahapatra Lily , Skinner Jason A. , Meylan Francoise , Chien Daniel , Dorward David W. , Chaussabel Damien , Siegel Richard M. , Nutman Thomas B.
Published: April 2014

Dysregulation of professional APC has been postulated as a major mechanism underlying Ag-specific T cell hyporesponsiveness in patients with patent filarial infection. To address the nature of this dysregulation, dendritic cells (DC) and macrophages generated from elutriated monocytes were exposed to live microfilariae (mf), the parasite stage that circulates in blood and is responsible for most immune dysregulation in filarial infections. DC exposed to mf for 24–96 h showed a marked increase in cell death and caspase-positive cells compared with unexposed DC, whereas mf exposure did not induce apoptosis in macrophages. Interestingly, 48-h exposure of DC to mf induced mRNA expression of the proapoptotic gene TRAIL and both mRNA and protein expression of TNF-α. mAb to TRAIL-R2, TNF-R1, or TNF-α partially reversed mf-induced cell death in DC, as did knocking down the receptor for TRAIL-R2 using small interfering RNA. The mf also induced gene expression of BH3-interacting domain death agonist and protein expression of cytochrome c in DC; mf-induced cleavage of BH3-interacting domain death agonist could be shown to induce release of cytochrome c, leading to activation of caspase 9. Our data suggest that mf induce DC apoptosis in a TRAIL- and TNF-α-dependent fashion.
Respiratory Epithelial Cells Convert Inactive Vitamin D to Its Active Form: Potential Effects on Host Defense
Authors: Hansdottir Sif , Monick Martha M. , Hinde Sara L. , Lovan Nina , Look Dwight C. , Hunninghake Gary W.
Published: April 2014

The role of vitamin D in innate immunity is increasingly recognized. Recent work has identified a number of tissues that express the enzyme 1α-hydroxylase and are able to activate vitamin D. This locally produced vitamin D is believed to have important immunomodulatory effects. In this paper, we show that primary lung epithelial cells express high baseline levels of activating 1α-hydroxylase and low levels of inactivating 24-hydroxylase. The result of this enzyme expression is that airway epithelial cells constitutively convert inactive 25-dihydroxyvitamin D3 to the active 1,25-dihydroxyvitamin D3. Active vitamin D that is generated by lung epithelium leads to increased expression of vitamin D-regulated genes with important innate immune functions. These include the cathelicidin antimicrobial peptide gene and the TLR coreceptor CD14. dsRNA increases the expression of 1α-hydroxylase, augments the production of active vitamin D, and synergizes with vitamin D to increase expression of cathelicidin. In contrast to induction of the antimicrobial peptide, vitamin D attenuates dsRNA-induced expression of the NF-κB-driven gene IL-8. We conclude that primary epithelial cells generate active vitamin D, which then influences the expression of vitamin D-driven genes that play a major role in host defense. Furthermore, the presence of vitamin D alters induction of antimicrobial peptides and inflammatory cytokines in response to viruses. These observations suggest a novel mechanism by which local conversion of inactive to active vitamin D alters immune function in the lung.
AspergillusConidia Activate the Complement by the Mannan-Binding Lectin C2 Bypass Mechanism
Authors: Dumestre-Pérard Chantal , Lamy Bertrand , Aldebert Delphine , Lemaire-Vieille Catherine , Grillot Renée , Brion Jean-Paul , Gagnon Jean , Cesbron Jean-Yves
Published: April 2014

Innate immunity is the major host defense against invasive aspergillosis. To determine whether the collectin mannan-binding lectin (MBL) is involved in the initial protective immunity through complement activation against opportunistic fungal infections caused by Aspergillus, we performed in vitro studies on 29 different strains of Aspergillus conidia from five different species. Incubation of Aspergillus conidia in human normal serum leads to activation of the alternative pathway, whereas neither the classical nor the lectin pathways through C4 and C2 cleavage are activated. Complement response to conidia was investigated using a MBL-deficient serum and reconstitution experiments were conducted with MBL/MASPs complexes. We found that MBL can directly support C3 activation by a C2 bypass mechanism. Finally, a stronger activation of the alternative pathway was observed for the clinical strains isolated from patients with invasive aspergillosis, compared with the environmental strains.
IL-15 Links TLR2/1-Induced Macrophage Differentiation to the Vitamin D-Dependent Antimicrobial Pathway
Authors: Krutzik Stephan R. , Hewison Martin , Liu Philip T. , Robles Juan Antonio , Stenger Steffen , Adams John S. , Modlin Robert L.
Published: April 2014

An essential function of the innate immune system is to directly trigger antimicrobial mechanisms to defend against invading pathogens. In humans, one such pathway involves activation by TLR2/1L leading to the vitamin D-dependent induction of antimicrobial peptides. In this study, we found that TLR2/1-induced IL-15 was required for induction of CYP27b1, the VDR and the downstream antimicrobial peptide cathelicidin. Although both IL-15 and IL-4 triggered macrophage differentiation, only IL-15 was sufficient by itself to induce CYP27b1 and subsequent bioconversion of 25-hydroxyvitamin D3 (25D3) into bioactive 1,25D3, leading to VDR activation and induction of cathelicidin. Finally, IL-15-differentiated macrophages could be triggered by 25D3 to induce an antimicrobial activity against intracellular Mycobacterium tuberculosis. Therefore, IL-15 links TLR2/1-induced macrophage differentiation to the vitamin D-dependent antimicrobial pathway.
A Novel Role for Neutrophils As Critical Activators of NK Cells
Authors: Spörri Roman , Joller Nicole , Hilbi Hubert , Oxenius Annette
Published: April 2014

Neutrophils are essential players in innate immune responses to bacterial infection. Despite the striking resistance of Legionella pneumophila (Lpn) to bactericidal neutrophil function, neutrophil granulocytes are important effectors in the resolution of legionellosis. Indeed, mice depleted of neutrophils were unable to clear Lpn due to a lack of the critical cytokine IFN-γ, which is produced by NK cells. We demonstrate that this can be ascribed to a previously unappreciated role of neutrophils as major NK cell activators. In response to Lpn infection, neutrophils activate caspase-1 and produce mature IL-18, which is indispensable for the activation of NK cells. Furthermore, we show that the IL-12p70 response in Lpn-infected neutropenic mice is also severely reduced and that the Lpn-induced IFN-γ production by NK cells is strictly dependent on IL-12. However, since dendritic cells, and not neutrophils, are the source of Lpn-induced IL-12, its paucity is a consequence of the absence of IFN-γ produced by NK cells rather than the absence of neutrophils per se. Therefore, neutrophil-derived IL-18, in combination with dendritic cell-produced IL-12, triggers IFN-γ synthesis in NK cells in Lpn-infected mice. We propose a novel central role for neutrophils as essential IL-18 producers and hence NK cell “helpers” in bacterial infection.
Species-Specific Interaction of Streptococcus pneumoniae with Human Complement Factor H
Authors: Lu Ling , Ma Zhuo , Jokiranta T. Sakari , Whitney Adeline R. , DeLeo Frank R. , Zhang Jing-Ren
Published: April 2014

Streptococcus pneumoniae naturally colonizes the nasopharynx as a commensal organism and sometimes causes infections in remote tissue sites. This bacterium is highly capable of resisting host innate immunity during nasopharyngeal colonization and disseminating infections. The ability to recruit complement factor H (FH) by S. pneumoniae has been implicated as a bacterial immune evasion mechanism against complement-mediated bacterial clearance because FH is a complement alternative pathway inhibitor. S. pneumoniae recruits FH through a previously defined FH binding domain of choline-binding protein A (CbpA), a major surface protein of S. pneumoniae. In this study, we show that CbpA binds to human FH, but not to the FH proteins of mouse and other animal species tested to date. Accordingly, deleting the FH binding domain of CbpA in strain D39 did not result in obvious change in the levels of pneumococcal bacteremia or virulence in a bacteremia mouse model. Furthermore, this species-specific pneumococcal interaction with FH was shown to occur in multiple pneumococcal isolates from the blood and cerebrospinal fluid. Finally, our phagocytosis experiments with human and mouse phagocytes and complement systems provide additional evidence to support our hypothesis that CbpA acts as a bacterial determinant for pneumococcal resistance to complement-mediated host defense in humans.