bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoimmunity to DNA and Nucleosomes in Binary Tetracycline-Regulated Polyomavirus T-Ag Transgenic Mice
Authors: Bendiksen Signy , Van Ghelue Marijke , Winkler Thomas , Moens Ugo , Rekvig Ole Petter
Published: April 2014

The mechanism(s) responsible for autoimmunity to DNA and nucleosomes in SLE is largely unknown. We have demonstrated that nucleosome-polyomavirus T-Ag complexes, formed in context of productive polyomavirus infection, activate dsDNA-specific B cells and nucleosome-specific CD4+ T cells. To investigate whether de novo expressed T-Ag is able to terminate nucleosome-specific T cell tolerance and to maintain anti-dsDNA Ab production in nonautoimmune mice, we developed two binary transgenic mouse variants in which expression of SV40 large T-Ag is controlled by tetracycline, MUP tTA/T-Ag (tet-off), and CMV rtTA/T-Ag (tet-on) mice. Data demonstrate that MUP tTA/T-Ag mice, but not CMV rtTA/T-Ag mice, are tightly controlling T-Ag expression. In MUP tTA/T-Ag transgenic mice, postnatal T-Ag expression activated CD8+ T cells but not DNA-specific B cells, while immunization with T-Ag and nucleosome-T-Ag-complexes before T-Ag expression resulted in elevated and remarkably stable titers of anti-T-Ag and anti-dsDNA Abs and activation of T-Ag-specific CD4+ T cells. Immunization of nonexpressing MUP tTA/T-Ag mice resulted in transient anti-T-Ag and anti-dsDNA Abs. This system reveals that a de novo expressed DNA-binding quasi-autoantigen maintain anti-dsDNA Abs and CD4+ T cell activation once initiated by immunization, demonstrating direct impact of a single in vivo expressed molecule on sustained autoimmunity to DNA and nucleosomes.
Atorvastatin Inhibits Autoreactive B Cell Activation and Delays Lupus Development in New Zealand Black/White F1 Mice
Authors: Lawman Sarah , Mauri Claudia , Jury Elizabeth C. , Cook H. Terrence , Ehrenstein Michael R.
Published: April 2014

Systemic lupus erythematosus is a multisystem autoimmune disease characterized by a wide range of immunological abnormalities that underlie the loss of tolerance. In this study we show that administration of atorvastatin to lupus-prone NZB/W F1 mice resulted in a significant reduction in serum IgG anti-dsDNA Abs and decreased proteinuria. Histologically, the treatment was associated with reduced glomerular Ig deposition and less glomerular injury. Disease improvement was paralleled by decreased expression of MHC class II on monocytes and B lymphocytes and reduced expression of CD80 and CD86 on B lymphocytes. Consequent upon this inhibition of Ag presentation, T cell proliferation was strongly impaired by atorvastatin in vitro and in vivo. A significant decrease in MHC class II expression was also observed in the target organ of lupus disease (i.e., the glomerulus). Serum cholesterol in atorvastatin-treated lupus mice fell to the level found in young NZB/W mice before disease onset. This is the first demonstration that atorvastatin can delay the progression of a spontaneous autoimmune disease and may specifically benefit patients with systemic lupus erythematosus.
T Cell Activation by Antibody-Like Immunoreceptors: Increase in Affinity of the Single-Chain Fragment Domain above Threshold Does Not Increase T Cell Activation against Antigen-Positive Target Cells but Decreases Selectivity
Authors: Chmielewski Markus , Hombach Andreas , Heuser Claudia , Adams Gregory P. , Abken Hinrich
Published: April 2014

Chimeric TCRs with an Ab-derived binding domain confer predefined specificity and MHC-independent target binding to T cells for use in adoptive immunotherapy. We investigated the impact of receptor binding affinity on the activation of grafted T cells. A series of anti-ErbB2 single-chain fragment binding domains with a Kd ranging from 3.2 × 10−7 to 1.5 × 10−11 M was linked to CD3ζ-derived immunoreceptors and expressed in human PBL. Solid phase bound ErbB2 protein triggered activation of receptor-grafted T cells in a dose-dependent manner. The activation threshold inversely correlated with the affinity of the receptor binding domain. The maximum level of cellular activation, however, was the same and independent of the binding affinity. Upon binding to ErbB2+ cells, T cells grafted with immunoreceptors carrying a single-chain fragment of Kd < 10−8 M were activated in a similar fashion against cells with different amounts of ErbB2 on the surface. T cells with a low affinity receptor (Kd > 10−8 M), however, were activated exclusively by cells with high amounts of ErbB2. In conclusion, recombinant immunoreceptors of higher affinity do not necessarily induce a more potent activation of T cells than low affinity immunoreceptors, but the higher affinity immunoreceptors exhibit less discrimination between target cells with high or low Ag expression levels.
Basal Igα/Igβ Signals Trigger the Coordinated Initiation of Pre-B Cell Antigen Receptor-Dependent Processes
Authors: Fuentes-Pananá Ezequiel M. , Bannish Gregory , Shah Neelima , Monroe John G.
Published: April 2014

The pro-B to pre-B transition during B cell development is dependent upon surface expression of a signaling competent pre-B cell Ag receptor (pre-BCR). Although the mature form of the BCR requires ligand-induced aggregation to trigger responses, the requirement for ligand-induced pre-BCR aggregation in promoting B cell development remains a matter of significant debate. In this study, we used transmission electron microscopy on murine primary pro-B cells and pre-B cells to analyze the aggregation state of the pre-BCR. Although aggregation can be induced and visualized following cross-linking by Abs to the pre-BCR complex, our analyses indicate that the pre-BCR is expressed on the surface of resting cells primarily in a nonaggregated state. To evaluate the degree to which basal signals mediated through nonaggregated pre-BCR complexes can promote pre-BCR-dependent processes, we used a surrogate pre-BCR consisting of the cytoplasmic regions of Igα/Igβ that is targeted to the inner leaflet of the plasma membrane of primary pro-B cells. We observed enhanced proliferation in the presence of low IL-7, suppression of VH(D)JH recombination, and induced κ light (L) chain recombination and cytoplasmic κ L chain protein expression. Interestingly, Igα/Igβ-mediated allelic exclusion was restricted to the B cell lineage as we observed normal TCRαβ expression on CD8-expressing splenocytes. This study directly demonstrates that basal signaling initiated through Igα/Igβ-containing complexes facilitates the coordinated control of differentiation events that are associated with the pre-BCR-dependent transition through the pro-B to pre-B checkpoint. Furthermore, these results argue that pre-BCR aggregation is not a requirement for pre-BCR function.
T Cell Immunity Induced by Live, Necrotic, and Apoptotic Tumor Cells
Authors: Bartholomae Wolf C. , Rininsland Frauke H. , Eisenberg Julia C. , Boehm Bernhard O. , Lehmann Paul V. , Tary-Lehmann Magdalena
Published: April 2014

The rules that govern the engagement of antitumor immunity are not yet fully understood. Ags expressed by tumor cells are prone to induce T cell tolerance unless the innate immune system is activated. It is unclear to what extent tumors engage this second signal link by the innate immune system. Apoptotic and necrotic (tumor) cells are readily recognized and phagocytosed by the cells of the innate immune system. It is unknown how this affects the tumor’s immunogenicity. Using a murine melanoma (B16m) and lymphoma (L5178Y-R) model, we studied the clonal sizes and cytokine signatures of the T cells induced by these tumors in syngeneic mice when injected as live, apoptotic, and necrotic cells. Both live tumors induced a type 2 CD4 cell response characterized by the prevalent production of IL-2, IL-4, and IL-5 over IFN-γ. Live, apoptotic, and necrotic cells induced CD4 (but no CD8) T cells of comparable frequencies and cytokine profiles. Therefore, live tumors engaged the second signal link, and apoptotic or necrotic tumor cell death did not change the magnitude or quality of the antitumor response. A subclone of L5178Y-R, L5178Y-S cells, were found to induce a high-frequency type 1 response by CD4 and CD8 cells that conveyed immune protection. The data suggest that the immunogenicity of tumors, and their characteristics to induce type 1 or type 2, CD4 or CD8 cell immunity is not primarily governed by signals associated with apoptotic or necrotic cell death, but is an intrinsic feature of the tumor itself.
A Tumor-Associated Glycoprotein That Blocks MHC Class II-Dependent Antigen Presentation by Dendritic Cells
Authors: Gutzmer Ralf , Li Wei , Sutterwala Shaheen , Lemos Maria P. , Elizalde J. Ignasi , Urtishak Sandra L. , Behrens Edward M. , Rivers Patricia M. , Schlienger Katia , Laufer Terri M. , Eck Stephen L. , Marks Michael S.
Published: April 2014

Tumors evade immune surveillance despite the frequent expression of tumor-associated Ags (TAA). Tumor cells escape recognition by CD8+ T cells through several mechanisms, including down-regulation of MHC class I molecules and associated Ag-processing machinery. However, although it is well accepted that optimal anti-tumor immune responses require tumor-reactive CD4+ T cells, few studies have addressed how tumor cells evade CD4+ T cell recognition. In this study, we show that a common TAA, GA733-2, and its murine orthologue, mouse epithelial glycoprotein (mEGP), function in blocking MHC class II-restricted Ag presentation by dendritic cells. GA733-2 is a common TAA that is expressed normally at low levels by some epithelial tissues and a subset of dendritic cells, but at high levels on colon, breast, lung, and some nonepithelial tumors. We show that ectopic expression of mEGP or GA733-2, respectively, in dendritic cells derived from murine bone marrow or human monocytes results in a dose-dependent inability to stimulate proliferation of Ag-specific or alloreactive CD4+ T cells. Dendritic cells exposed to cell debris from tumors expressing mEGP are similarly compromised. Furthermore, mice immunized with dendritic cells expressing mEGP from a recombinant adenovirus vector exhibited a muted anti-adenovirus immune response. The inhibitory effect of mEGP was not due to down-regulation of functional MHC class II molecules or active suppression of T cells, and did not extend to T cell responses to superantigen. These results demonstrate a novel mechanism by which tumors may evade CD4+ T cell-dependent immune responses through expression of a TAA.
Specific Engagement of TLR4 or TLR3 Does Not Lead to IFN-β-Mediated Innate Signal Amplification and STAT1 Phosphorylation in Resident Murine Alveolar Macrophages
Authors: Punturieri Antonello , Alviani Rebecca S. , Polak Timothy , Copper Phil , Sonstein Joanne , Curtis Jeffrey L.
Published: April 2014

The innate immune response must be mobilized promptly yet judiciously via TLRs to protect the lungs against pathogens. Stimulation of murine peritoneal macrophage (PMφ) TLR4 or TLR3 by pathogen-associated molecular patterns (PAMPs) typically induces type I IFN-β, leading to autocrine activation of the transcription factor STAT1. Because it is unknown whether STAT1 plays a similar role in the lungs, we studied the response of resident alveolar macrophages (AMφ) or control PMφ from normal C57BL/6 mice to stimulation by PAMPs derived from viruses (polyriboinosinic:polyribocytidylic acid, specific for TLR3) or bacteria (Pam3Cys, specific for TLR2, and repurified LPS, specific for TLR4). AMφ did not activate STAT1 by tyrosine phosphorylation on Y701 following stimulation of any of these three TLRs, but readily did so in response to exogenous IFN-β. This unique AMφ response was not due to altered TLR expression, or defective immediate-early gene response, as measured by expression of TNF-α and three β chemokines. Instead, AMφ differed from PMφ in not producing bioactive IFN-β, as confirmed by ELISA and by the failure of supernatants from TLR-stimulated AMφ to induce STAT1 phosphorylation in PMφ. Consequently, AMφ did not produce the microbicidal effector molecule NO following TLR4 or TLR3 stimulation unless exogenous IFN-β was also added. Thus, murine AMφ respond to bacterial or viral PAMPs by producing inflammatory cytokines and chemokines, but because they lack the feed-forward amplification typically mediated by autocrine IFN-β secretion and STAT1 activation, require exogenous IFN to mount a second phase of host defense.
Oral Tolerance to Nickel Requires CD4+ Invariant NKT Cells for the Infectious Spread of Tolerance and the Induction of Specific Regulatory T Cells
Authors: Roelofs-Haarhuis Karin , Wu Xianzhu , Gleichmann Ernst
Published: April 2014

Previously, oral administration of nickel to C57BL/6 wild-type (WT) mice was shown to render both their splenic T cells and APCs (i.e., T cell-depleted spleen cells) capable of transferring nickel tolerance to naive syngeneic recipients. Moreover, sequential adoptive transfer experiments revealed that on transfer of tolerogenic APCs and immunization, the naive T cells of the recipients differentiated into regulatory T (Treg) cells. Here, we demonstrate that after oral nickel treatment Jα18−/− mice, which lack invariant NKT (iNKT) cells, were not tolerized and failed to generate Treg cells. However, transfer of APCs from those Jα18−/− mice did tolerize WT recipients. Hence, during oral nickel administration, tolerogenic APCs are generated that require iNKT cell help for the induction of Treg cells. To obtain this help, the tolerogenic APCs must address the iNKT cells in a CD1-restricted manner. When Jα18−/− mice were used as recipients of cells from orally tolerized WT donors, the WT Treg cells transferred the tolerance, whereas WT APCs failed to do so, although they proved tolerogenic on transfer to WT recipients. However, Jα18−/− recipients did become susceptible to the tolerogenicity of transferred WT APCs when they were reconstituted with IL-4- and IL-10-producing CD4+ iNKT cells. We conclude that CD4+ iNKT cells are required for the induction of oral nickel tolerance and, in particular, for the infectious spread of tolerance from APCs to T cells. Once induced, these Treg cells, however, can act independently of iNKT cells.
Requirements of NK Cells and Proinflammatory Cytokines in T Cell-Dependent Neonatal Autoimmune Ovarian Disease Triggered by Immune Complex
Authors: Setiady Yulius Y. , Pramoonjago Patcharin , Tung Kenneth S. K.
Published: April 2014

A model of neonatal autoimmune disease has been described recently in which an epitope-specific autoantibody to murine zona pellucida 3 induces severe ovarian disease in neonatal, but not adult, mice (neonatal AOD). The autoantibody forms immune complex with endogenous ovarian zona pellucida 3, and a pathogenic CD4+ T cell response is triggered. The basis for the predominant neonatal susceptibility has not been clarified. In this study innate immunity, including neonatal NK cells, in neonatal AOD was investigated. Neonatal spleen contained readily detectable NK1.1+TCRVβ−, but not NK1.1+TCRVβ+, cells. Ab depletion of NK1.1+TCRVβ− cells inhibited neonatal AOD development. Moreover, in adoptive transfer of neonatal AOD, recipient disease was ameliorated when either donor or recipient NK cells were depleted. Thus, NK cells operate in both induction and effector phases of the disease. IFN-γ was produced by neonatal NK cells in vivo, and it may be important in neonatal AOD. Indeed, ovaries with neonatal AOD expressed high levels of IFN-γ and TNF-α which correlated with disease severity, and the disease was inhibited by IFN-γ or TNF-α Ab. Importantly, disease was enhanced by recombinant IFN-γ, and treatment of T cell donors with IFN-γ Ab also significantly reduced adoptive transfer of neonatal AOD. Finally, neonatal AOD was ameliorated in mice deficient in FcγRIII and was enhanced in FcγRIIB-deficient mice. We conclude that neonatal NK cells promote pathogenic T cell response at multiple stages during neonatal autoimmune disease pathogenesis. Also operative in neonatal AOD are other mediators of the innate system, including proinflammatory cytokines and FcγRIII signaling.
Resistance to CD4+CD25+ Regulatory T Cells and TGF-β in Cbl-b−/− Mice
Authors: Wohlfert Elizabeth A. , Callahan Margaret K. , Clark Robert B.
Published: April 2014

Cbl-b−/− mice have signaling defects that result in CD28-independent T cell activation, increased IL-2 production, hyper-reactive T cells, and increased autoimmunity. Although the increased autoimmunity in these mice is believed to result from the hyper-reactive T cells, the mechanisms leading from T cell hyper-reactivity to autoimmunity remain unclear. Specifically, the function and interaction of CD4+CD25+ regulatory T cells (Treg) and CD4+CD25− effector T cells (Teff) in Cbl-b−/− mice have not been examined. We now report that Cbl-b−/− CD4+CD25+ Treg exhibit normal regulatory function in vitro. In contrast, the in vitro response of Cbl-b−/− CD4+CD25− Teff is abnormal, in that it is not inhibited by either Cbl-b−/− or wild-type Treg. This resistance of Cbl-b−/− Teff to in vitro regulation is seen at the levels of both DNA synthesis and cell division. In addition to this resistance to CD4+CD25+ Treg, Cbl-b−/− Teff demonstrate in vitro resistance to inhibition by TGF-β. This second form of resistance in Cbl-b−/− Teff is seen despite the expression of normal levels of type II TGF-β receptors and normal levels of phosphorylated Smad3 after TGF-β stimulation. Coupled with recent reports of resistance to Treg in Teff exposed to LPS-treated dendritic cells, our present findings suggest that resistance to regulation may be a relevant mechanism in both normal immune function and autoimmunity.
TNF Activates Syk Protein Tyrosine Kinase Leading to TNF-Induced MAPK Activation, NF-κB Activation, and Apoptosis
Authors: Takada Yasunari , Aggarwal Bharat B.
Published: April 2014

Spleen tyrosine kinase (Syk), a nonreceptor protein kinase initially found to be expressed only in hemopoietic cells, has now been shown to be expressed in nonhemopoietic cells and to mediate signaling of various cytokines. Whether Syk plays any role in TNF signaling was investigated. Treatment of Jurkat T cells with TNF activated Syk kinase but not ZAP70, another member of Syk kinase family, and the optimum activation occurred at 10 s and with 1 nM TNF. TNF also activated Syk in myeloid and epithelial cells. TNF-induced Syk activation was abolished by piceatannol (Syk-selective inhibitor), which led to the suppression of TNF-induced activation of c- JNK, p38 MAPK, and p44/p42 MAPK. Jurkat cells that did not express Syk (JCaM1, JCaM1/lck) showed lack of TNF-induced Syk, JNK, p38 MAPK, and p44/p42 MAPK activation, as well as TNF-induced IκBα phosphorylation, IκBα degradation, and NF-κB activation. TNF-induced NF-κB activation was enhanced by overexpression of Syk by Syk-cDNA and suppressed when Syk expression was down-regulated by expression of Syk-small interfering RNA (siRNA-Syk). The apoptotic effects of TNF were reduced by up-regulation of NF-κB by Syk-cDNA, and enhanced by down-regulation of NF-κB by siRNA-Syk. Immunoprecipitation of cells with Syk Abs showed TNF-dependent association of Syk with both TNFR1 and TNFR2; this association was enhanced by up-regulation of Syk expression with Syk-cDNA and suppressed by down-regulation of Syk using siRNA-Syk. Overall, our results demonstrate that Syk activation plays an essential role in TNF-induced activation of JNK, p38 MAPK, p44/p42 MAPK, NF-κB, and apoptosis.
Two Human ULBP/RAET1 Molecules with Transmembrane Regions Are Ligands for NKG2D
Authors: Bacon Louise , Eagle Robert A. , Meyer Martina , Easom Nicholas , Young Neil T. , Trowsdale John
Published: April 2014

We characterized two novel members of the RAET1/ULBP gene cluster, RAET1E and RAET1G. The encoded proteins were similar to the ULBP in their class I-like α1 and α2 domains, but differed in that, instead of being GPI-anchored, their sequences were type 1 membrane-spanning molecules. Both proteins were capable of being expressed at the cell surface. Both proteins bound the activating receptor NKG2D, and RAET1G bound the human CMV protein UL16. The expression of diverse NKG2D-binding molecules in different tissues and with different properties is consistent with multiple modes of infection- or stress-induced activation.
Dissection of the HLA-DR4 Peptide Repertoire in Endocrine Epithelial Cells: Strong Influence of Invariant Chain and HLA-DM Expression on the Nature of Ligands
Authors: Muntasell Aura , Carrascal Montserrat , Alvarez Iñaki , Serradell Laurence , van Veelen Peter , Verreck Frank A. W. , Koning Frits , Abian Joaquín , Jaraquemada Dolores
Published: April 2014

Class II MHC (MHC II) expression is restricted to professional APCs and thymic epithelium but it also occurs in the epithelial cells of autoimmune organs which are the unique targets of the CD4 autoreactive T cells in endocrine autoimmune diseases. This specificity is presumably conditioned by an epithelium-specific peptide repertoire associated to MHC II at the cell surface. MHC II expression and function is dependent on the action of two main chaperones, invariant chain (Ii) and DM, whose expression is coregulated with MHC II. However, there is limited information about the in vivo expression levels of these molecules and uncoordinated expression has been demonstrated in class II-positive epithelial cells that may influence the MHC-associated peptide repertoires and the outcome of the autoimmune response. We have examined the pool of peptides associated to DR4 molecules expressed by a neuroendocrine epithelial cell and the consequences of Ii and DM coexpression. The RINm5F rat insulinoma cell line was transfected with HLA-DRB1*0401, Ii, and DM molecules in four different combinations: RIN-DR4, -DR4Ii, -DR4DM, and -DR4IiDM. The analysis of the peptide repertoire and the identification of the DR4 naturally processed ligands in each transfected cell were achieved by mass spectrometry. The results demonstrate that 1) the expression of Ii and DM affected the DR4 peptide repertoires by producing important variations in their content and in the origin of peptides; 2) these restrictions affected the stability and sequence of the peptides of each repertoire; and 3) Ii and DM had both independent and coordinate effects on these repertoires.
Analysis of Transcription Factor Expression during Discrete Stages of Postnatal Thymocyte Differentiation
Authors: Tabrizifard Sahba , Olaru Alexandru , Plotkin Jason , Fallahi-Sichani Mohammad , Livak Ferenc , Petrie Howard T.
Published: April 2014

Postnatal T lymphocyte differentiation in the thymus is a multistage process involving serial waves of lineage specification, proliferative expansion, and survival/cell death decisions. Although these are believed to originate from signals derived from various thymic stromal cells, the ultimate consequence of these signals is to induce the transcriptional changes that are definitive of each step. To help to characterize this process, high density microarrays were used to analyze transcription factor gene expression in RNA derived from progenitors at each stage of T lymphopoietic differentiation, and the results were validated by a number of appropriate methods. We find a large number of transcription factors to be expressed in developing T lymphocytes, including many with known roles in the control of differentiation, proliferation, or cell survival/death decisions in other cell types. Some of these are expressed throughout the developmental process, whereas others change substantially at specific developmental transitions. The latter are particularly interesting, because stage-specific changes make it increasingly likely that the corresponding transcription factors may be involved in stage-specific processes. Overall, the data presented here represent a large resource for gene discovery and for confirmation of results obtained through other methods.
STAT1 Regulates Lipopolysaccharide- and TNF-α-Dependent Expression of Transporter Associated with Antigen Processing 1 and Low Molecular Mass Polypeptide 2 Genes in Macrophages by Distinct Mechanisms
Authors: Marqués Laura , Brucet Marina , Lloberas Jorge , Celada Antonio
Published: April 2014

Transporter associated with Ag processing 1 and low molecular mass polypeptide 2 (LMP2) are essential for class I MHC function and share a common bidirectional promoter. In murine bone marrow-derived macrophages, LPS and TNF-α induced Tap1 and up-regulated Lmp2, which is constitutively expressed at low levels. These two genes are induced by LPS and TNF-α with distinct kinetics, at 6 and 12–24 h, respectively. Using macrophages derived from the TNF-α receptors of knockout mice, we found that induction by LPS is not due to the autocrine production of TNF-α. In macrophages from STAT-1 knockout mice, neither LPS nor TNF-α induced the expression of Tap1 or Lmp2. The shared promoter contains several areas that can be controlled by STAT-1, such as the proximal and distal IFN-γ activation site (GAS) boxes in the direction of the Tap1 gene. By making deletions of the promoter, we determined that only the proximal GAS box is required for LPS induction of Tap1 and Lmp2. In contrast, TNF-α induction of these two genes is dependent on the IFN regulatory factor-1 and NF-κB boxes, and not on the GAS box. Our experiments using gel shift analysis and Abs indicated that STAT1 binds to the GAS box in nuclear extracts from LPS-treated macrophages. The nuclear extracts obtained from macrophages treated with TNF-α bound to the IFN regulatory factor-1 and NF-κB boxes. These results show that LPS and TNF-α regulate the induction of Tap1 and Lmp2 through STAT1, but use distinct areas of the promoter.
The E2F-1 Transcription Factor Promotes Caspase-8 and Bid Expression, and Enhances Fas Signaling in T Cells
Authors: Cao Qingyu , Xia Ying , Azadniv Mitra , Crispe I. Nicholas
Published: April 2014

The immune system depends on the extensive proliferation of rare Ag-specific precursor T lymphocytes, followed by their differentiation, the delivery of effector function, and finally death by apoptosis. T cells that lack the E2F-1 transcription factor, which is activated as cells pass the restriction point and enter S phase, show defects in activation-induced cell death. We now report that E2F-1 increases the activity of an apoptotic pathway that is important in murine primary T cells. Thus, E2F-1 promotes the transcription of Bid, a molecule that links death receptor signaling to the activation of apoptotic mechanisms in mitochondria. It also promotes the transcription of caspase-8, the enzyme that cleaves and activates Bid. Enforced expression of Bid can partially restore apoptosis in E2F-1-deficient T cells. Thus, E2F-1 integrates cell cycle progression with apoptosis.
A Short Consensus Repeat-Containing Complement Regulatory Protein of Lamprey That Participates in Cleavage of Lamprey Complement 3
Authors: Kimura Yuko , Inoue Naokazu , Fukui Aya , Oshiumi Hiroyuki , Matsumoto Misako , Nonaka Mayumi , Kuratani Shigeru , Fujita Teizo , Nonaka Masaru , Seya Tsukasa
Published: April 2014

The prototype of the short consensus repeat (SCR)-containing C regulatory protein is of interest in view of its evolutionary significance with regard to the origin of the C regulatory system. Lamprey is an agnathan fish that belongs to the lowest class of vertebrates. Because it does not possess lymphocytes, it lacks Ig and consequently the classical C pathway. We identified an SCR-containing C regulatory protein from the lamprey. The primary structure predicted from the cDNA sequence showed that this is a secretary protein consisting of eight SCRs. This framework is similar to the α-chain of C4b-binding protein (C4bp). SCR2 and -3 of human C4bp are essential for C4b inactivation, and this region is fairly well conserved in the lamprey protein. However, the other SCRs of this protein are similar to those of other human C regulatory proteins. The lamprey protein binds to the previously reported lamprey C3b/C3bi deposited on yeast and cleaves lamprey C3b-like C3 together with a putative serum protease. The scheme resembles the C regulatory system of mammals, where factor I and its cofactor inactivate C3b. Unlike human cofactors, the lamprey protein requires divalent cations for C3b-like C3 cleavage. Its artificial membrane-anchored form protects host cells from lamprey C attack via the lectin pathway. Thus, the target of this protein appears to be C3b and/or its family. We named this protein Lacrep, the lamprey C regulatory protein. Lacrep is a member of SCR-containing C regulators, the first of its kind identified in the lowest vertebrates.
Unprecedented Multiplicity of Ig Transmembrane and Secretory mRNA Forms in the Cartilaginous Fish
Authors: Rumfelt Lynn L. , Diaz Marilyn , Lohr Rebecca L. , Mochon Evonne , Flajnik Martin F.
Published: April 2014

In most jawed vertebrates including cartilaginous fish, membrane-bound IgM is expressed as a five Ig superfamily (Igsf)-domain H chain attached to a transmembrane (Tm) region. Heretofore, bony fish IgM was the one exception with IgM mRNA spliced to produce a four-domain Tm H chain. We now demonstrate that the Tm and secretory (Sec) mRNAs of the novel cartilaginous fish Ig isotypes, IgW and IgNAR, are present in multiple forms, most likely generated by alternative splicing. In the nurse shark, Ginglymostoma cirratum, and horn shark, Heterodontus francisci, alternative splicing of Tm exons to the second or the fourth constant (CH) exons produces two distinct IgW Tm cDNAs. Although the seven-domain IgW Sec cDNA form contains a canonical secretory tail shared with IgM, IgNAR, and IgA, we report a three-domain cDNA form of shark IgW (IgWshort) having an unusual Sec tail, which is orthologous to skate IgXshort cDNA. The IgW and IgWshort Sec transcripts are restricted in their tissue distribution and expression levels vary among individual sharks, with all forms expressed early in ontogeny. IgNAR mRNA is alternatively spliced to produce a truncated four-domain Tm cDNA and a second Tm cDNA is expressed identical in Igsf domains as the Sec form. PBL is enriched in the Tm cDNA of these Igs. These molecular data suggest that cartilaginous fish have augmented their humoral immune repertoire by diversifying the sizes of their Ig isotypes. Furthermore, these Tm cDNAs are prototypical and the truncated variants may translate as more stable protein at the cell surface.
Peptide Variants of Viral CTL Epitopes Mediate Positive Selection and Emigration of Ag-Specific Thymocytes In Vivo
Authors: Fridkis-Hareli Masha , Reche Pedro A. , Reinherz Ellis L.
Published: April 2014

During development, thymocytes carrying TCRs mediating low-affinity interactions with MHC-bound self-peptides are positively selected for export into the mature peripheral T lymphocyte pool. Thus, exogenous administration of certain altered peptide ligands (APL) with reduced TCR affinity relative to cognate Ags may provide a tool to elicit maturation of desired TCR specificities. To test this “thymic vaccination” concept, we designed APL of the viral CTL epitopes gp33–41 and vesicular stomatitis virus nucleoprotein octapeptide N52–59 relevant for the lymphocytic choriomeningitis virus-specific P14- and vesicular stomatitis virus-specific N15-TCRs, respectively, and examined their effects on thymocytes in vivo using irradiation chimeras. Injection of APL into irradiated congenic (Ly-5.1) mice, reconstituted with T cell progenitors from the bone marrow of P14 RAG2−/− (Ly-5.2) or N15 RAG2−/− (Ly-5.2) transgenic mice, resulted in positive selection of T cells expressing the relevant specificity. Moreover, the variants led to export of virus-specific T cells to lymph nodes, but without inducing T cell proliferation. These findings show that the mature T cell repertoire can be altered by in vivo peptide administration through manipulation of thymic selection.
Tyrosine Kinase 2 Interacts with and Phosphorylates Receptor for Activated C Kinase-1, a WD Motif-Containing Protein
Authors: Haro Takashi , Shimoda Kazuya , Kakumitsu Haruko , Kamezaki Kenjirou , Numata Akihiko , Ishikawa Fumihiko , Sekine Yuichi , Muromoto Ryuta , Matsuda Tadashi , Harada Mine
Published: April 2014

Receptor for activated C kinase (Rack)-1 is a protein kinase C-interacting protein, and contains a WD repeat but has no enzymatic activity. In addition to protein kinase C, Rack-1 also binds to Src, phospholipase Cγ, and ras-GTPase-activating proteins. Thus, Rack-1 is thought to function as a scaffold protein that recruits specific signaling elements. In a cytokine signaling cascade, Rack-1 has been reported to interact with the IFN-αβ receptor and Stat1. In addition, we show here that Rack-1 associates with a member of Jak, tyrosine kinase 2 (Tyk2). Rack-1 interacts weakly with the kinase domain and interacts strongly with the pseudokinase domain of Tyk2. Rack-1 associates with Tyk2 via two regions, one in the N terminus and one in the middle portion (aa 138–203) of Rack-1. Jak activation causes the phosphorylation of tyrosine 194 on Rack-1. After phosphorylation, Rack-1 is translocated toward the perinuclear region. In addition to functioning as a scaffolding protein, these results raise the possibility that Rack-1 functions as a signaling molecule in cytokine signaling cascades.
Direct Repression of prdm1 by Bcl-6 Inhibits Plasmacytic Differentiation
Authors: Tunyaplin Chainarong , Shaffer A. L. , Angelin-Duclos Cristina D. , Yu Xin , Staudt Louis M. , Calame Kathryn L.
Published: April 2014

We have identified two intronic regions of mouse prdm1, the gene encoding B lymphocyte-induced maturation protein-1 (Blimp-1), which confer transcriptional repression in response to Bcl-6. The Bcl-6 response element in intron 5, which is conserved between mice and humans, was studied in detail. It binds Bcl-6 in vitro and was shown by chromatin immunoprecipitation to be occupied by Bcl-6 in vivo. Neither Bcl-6 response element functions as a STAT3-response element, showing that STAT3 does not compete with Bcl-6 at these sites. Bcl-6−/− mice confirm the biological importance of Bcl-6-dependent repression of prdm1. These mice have elevated Ab response, increased Ig-secreting cells, and increased Blimp-1+ cells in spleen following immunization and their splenic B cells show accelerated plasmacytic development in vitro.
TLR4 Is the Signaling but Not the Lipopolysaccharide Uptake Receptor
Authors: Dunzendorfer Stefan , Lee Hyun-Ku , Soldau Katrin , Tobias Peter S.
Published: April 2014

TLR4 is the primary recognition molecule for inflammatory responses initiated by bacterial LPS (endotoxin). Internalization of endotoxin by various cell types is an important step for its removal and detoxification. Because of its role as an LPS-signaling receptor, TLR4 has been suggested to be involved in cellular LPS uptake as well. LPS uptake was investigated in primary monocytes and endothelial cells derived from TLR4 and CD14 knockout C57BL/6 mice using tritiated and fluorescein-labeled LPS. Intracellular LPS distribution was investigated by deconvolution confocal microscopy. We could not observe any difference in LPS uptake and intracellular LPS distribution in either monocytes or endothelial cells between TLR4−/− and wild-type cells. As expected, CD14−/− monocytes showed a highly impaired LPS uptake, confirming CD14-dependent uptake in monocytes. Upon longer incubation periods, the CD14-deficient monocytes mimicked the LPS uptake pattern of endothelial cells. Endothelial cell LPS uptake is slower than monocyte uptake, LBP rather than CD14 dependent, and sensitive to polyanionic polymers, which have been shown to block scavenger receptor-dependent uptake mechanisms. We conclude that TLR4 is not involved in cellular LPS uptake mechanisms. In membrane CD14-positive cells, LPS is predominantly taken up via CD14-mediated pathways, whereas in the CD14-negative endothelial cells, there is a role for scavenger receptor-dependent pathways.
Suppression of Ongoing Adjuvant-Induced Arthritis by Neutralizing the Function of the p28 Subunit of IL-27
Authors: Goldberg Ruth , Wildbaum Gizi , Zohar Yaniv , Maor Gila , Karin Nathan
Published: April 2014

IL-27 is a recently defined family member of the long-chain four-helix bundle cytokines, which consists of EBI3, an IL-12p40-related protein, and p28, an IL-12p35-related polypeptide. The role of IL-27 in the regulation of inflammatory autoimmune diseases has never been studied. The current study uses the DNA vaccination technology, and highly specific Abs to the p28 subunit of IL-27 that were generated by this technology, to delineate its role in the regulation of adjuvant-induced arthritis in Lewis rats. Neutralizing the in vivo function of IL-27 by targeted DNA vaccines and by Abs against IL-27 p28 that were produced in protected donors could rapidly suppress an ongoing disease. Disease suppression was associated with a reduced ex vivo production of inflammatory cytokines. We then used these Abs to investigate the mechanistic basis of disease suppression, showing that IL-27 is not only involved in directing the polarization of naive T cells, but also affects the proliferative response and cytokine production of Ag-specific effector/memory Th1 cells. This may explain, in part, its important role in the regulation of inflammatory autoimmune diseases, and also suggest novel ways of therapy.
TLR9 Is Localized in the Endoplasmic Reticulum Prior to Stimulation
Authors: Leifer Cynthia A. , Kennedy Margaret N. , Mazzoni Alessandra , Lee ChangWoo , Kruhlak Michael J. , Segal David M.
Published: April 2014

In mammals, 10 TLRs recognize conserved pathogen-associated molecular patterns, resulting in the induction of inflammatory innate immune responses. One of these, TLR9, is activated intracellularly by bacterial DNA and synthetic oligodeoxynucleotides (ODN), containing unmethylated CpG dinucleotides. Following treatment with CpG ODN, TLR9 is found in lysosome-associated membrane protein type 1-positive lysosomes, and we asked which intracellular compartment contains TLR9 before CpG exposure. Surprisingly, we found by microscopy and supporting biochemical evidence that both transfected and endogenously expressed human TLR9 is retained in the endoplasmic reticulum. By contrast, human TLR4 trafficked to the cell surface, indicating that endoplasmic reticulum retention is not a property common to all TLRs. Because TLR9 is observed in endocytic vesicles following exposure to CpG ODN, our data indicate that a special mechanism must exist for translocating TLR9 to the signaling compartments that contain the CpG DNA.
Role of IFN Regulatory Factor-1 and IL-12 in Immunological Resistance to Pathogenesis of N-Methyl-N-Nitrosourea-Induced T Lymphoma
Authors: Liu Jianguo , Xiang Zhaoying , Ma Xiaojing
Published: April 2014

IFN regulatory factor-1 (IRF-1) is a critical effector molecule in IFN signaling and acts as a tumor suppressor and tumor susceptibility gene. IL-12 is a key factor in the induction of innate resistance and generation of Th1 cells and CTL. Our recent study has revealed an intimate relationship between IRF-1 and IL-12 in that IRF-1 regulates the production of IL-12 by selectively controlling transcriptional activation of IL-12 p35 gene. In this work, we find that IRF-1-deficient mice are highly susceptible to N-methyl-N-nitrosourea (MNU)-induced T lymphomas. This susceptibility is associated with strong defects in the expression of IL-12, lymphotoxin (LT)β, and IFN-γ. Consistently, IL-12 p35−/−, IFN-γ−/−, and LTβ−/− mice are also highly vulnerable to MNU-induced carcinogenesis. Administration of rIL-12 to IRF-1−/− mice restores normal expression of LTβ and IFN-γ, and significantly enhances the ability of IRF-1−/− mice to resist MNU-induced pathogenesis. This strongly suggests an IRF-1/IL-12/IFN-γ regulatory axis in tumor surveillance. By DNA microarray analysis, we comprehensively identify differences and patterns in gene expression in splenocytes of wild-type (WT) vs IRF-1−/− mice challenged with MNU. This study contributes to efforts to elucidate the cellular/molecular mechanisms and the downstream players involved in IRF-1-mediated host defense against lymphoproliferative malignancies.
Engagement of the Pathogen Survival Response Used by Group A Streptococcus to Avert Destruction by Innate Host Defense
Authors: Voyich Jovanka M. , Braughton Kevin R. , Sturdevant Daniel E. , Vuong Cuong , Kobayashi Scott D. , Porcella Stephen F. , Otto Michael , Musser James M. , DeLeo Frank R.
Published: April 2014

Neutrophils are a critical component of human innate host defense and efficiently kill the vast majority of invading microorganisms. However, bacterial pathogens such as group A Streptococcus (GAS) successfully avert destruction by neutrophils to cause human infections. Relatively little is known about how pathogens detect components of the innate immune system to respond and survive within the host. In this study, we show that inactivation of a two-component gene regulatory system designated Ihk-Irr significantly attenuates streptococcal virulence in mouse models of soft tissue infection and bacteremia. Microarray analysis of wild-type and irr-negative mutant (irr mutant) GAS strains revealed that Ihk-Irr influenced expression of 20% of all transcripts in the pathogen genome. Notably, at least 11 genes involved in cell wall synthesis, turnover, and/or modification were down-regulated in the irr mutant strain. Compared with the wild-type strain, significantly more of the irr mutant strain was killed by human neutrophil components that destroy bacteria by targeting the cell envelope (cell wall and/or membrane). Unexpectedly, expression of ihk and irr was dramatically increased in the wild-type strain exposed to these same neutrophil products under conditions that favored cell envelope damage. We report a GAS mechanism for detection of innate host defense that initiates the pathogen survival response, in which cell wall synthesis is critical. Importantly, our studies identify specific genes in the pathogen survival response as potential targets to control human infections.
Secretion of Intracellular IL-1 Receptor Antagonist (Type 1) Is Dependent on P2X7 Receptor Activation
Authors: Wilson Heather L. , Francis Sheila E. , Dower Steven K. , Crossman David C.
Published: April 2014

Inflammatory mechanisms are critical in the arterial response to injury. Both IL-1 and the naturally occurring inhibitor of IL-1, IL-1R antagonist (IL-1ra), are expressed in the arterial wall, and in particular in the endothelium. Previous studies suggest that endothelial cells only make the intracellular type I isoform of IL-1ra (icIL-1ra1), an isoform known to lack a secretory signal peptide. It is unclear how icIL-1ra is released from the endothelial cell to act as an antagonist on cell surface IL-1 type I receptors. IL-1β, which also lacks a secretory signal peptide, may be released by ATP stimulation of the P2X7R. Therefore, we examined whether icIL-1ra1 release occurs in an analogous manner, using both the mouse macrophage cell line RAW264.7 and HUVECs. P2X7R activation caused icIL-1ra1 release from LPS-primed RAW264.7 macrophages and from HUVECs. This release was inhibited in the absence of extracellular calcium, and attenuated by preincubation with oxidized ATP, KN62, and apyrase. Endogenous ATP release, which also facilitated release of icIL-1ra1, was detected during LPS treatment of both RAW264.7 macrophages and HUVECs. Annexin V assays showed that ATP stimulation resulted in a rapid phosphatidylserine (PS) exposure on the cell surface of RAW264.7 macrophages, and that PS-exposed microvesicles contained icIL-1ra1. However, PS flip and microvesicle shedding was not apparent in ATP-treated HUVECs. These data support a general role for the P2X7R in the release of leaderless cytokines into the extracellular medium, and indicate how icIL-1ra1 may act upon its extracellular target, the IL-1R.
Activation, Differentiation, and Migration of Naive Virus-Specific CD8+ T Cells during Pulmonary Influenza Virus Infection
Authors: Lawrence Christopher W. , Braciale Thomas J.
Published: April 2014

The low precursor frequency of individual virus-specific CD8+ T cells in a naive host makes the early events of CD8+ T cell activation, proliferation, and differentiation in response to viral infection a challenge to identify. We have therefore examined the response of naive CD8+ T cells to pulmonary influenza virus infection with a murine adoptive transfer model using hemagglutinin-specific TCR transgenic CD8+ T cells. Initial activation of CD8+ T cells occurs during the first 3 days postinfection exclusively within the draining lymph nodes. Acquisition of CTL effector functions, including effector cytokine and granule-associated protease expression, occurs in the draining lymph nodes and differentially correlates with cell division. Division of activated CD8+ T cells within the draining lymph nodes occurs in an asynchronous manner between days 3 and 4 postinfection. Despite the presence of Ag for several days within the draining lymph nodes, dividing T cells do not appear to maintain contact with residual Ag. After multiple cell divisions, CD8+ T cells exit the draining lymph nodes and migrate to the infected lung. Activated CD8+ T cells also disseminate throughout lymphoid tissue including the spleen and distal lymph nodes following their emigration from draining lymph nodes. These results demonstrate an important role for draining lymph nodes in orchestrating T cell responses during a local infection of a discrete organ to generate effector CD8+ T cells capable of responding to infection and seeding peripheral lymphoid tissues.
Microbial DNA Induces a Host Defense Reaction of Human Respiratory Epithelial Cells
Authors: Platz Juliane , Beisswenger Christoph , Dalpke Alexander , Koczulla Rembert , Pinkenburg Olaf , Vogelmeier Claus , Bals Robert
Published: April 2014

Epithelial cells represent the initial site of bacterial colonization in the respiratory tract. TLR9 has been identified in B cells and CD 123+ dendritic cells and found to be involved in the recognition of microbial DNA. It was the aim of the study to investigate the role of TLR9 in the host defense reactions of the respiratory epithelium. Respiratory epithelial cell lines (IHAEo−, Calu-3) or fully differentiated primary human cells as air-liquid interface cultures were stimulated with bacterial DNA or synthetic oligonucleotides containing CpG motifs (CpG oligodeoxynucleotides). Expression of TLR9, cytokines, and human β-defensin 2 was determined by quantitative RT-PCR or by ELISA. We found that TLR9 is expressed by respiratory epithelial cell lines and fully differentiated primary epithelial cells at low levels. Stimulation of the above-mentioned cells with bacterial DNA or CpG oligodeoxynucleotide resulted in an inflammatory reaction characterized by a dose-dependent up-regulation of cytokines (IL-6, IL-8) and human β-defensin 2. Up-regulation of NF-κB in epithelial cells in response to the CpG motif containing DNA was inhibited by overexpression of a dominant negative form of MyD88. These results provide clear evidence that the human respiratory epithelium is capable of detecting microbial DNA by TLR9. The respiratory epithelium has an important function in triggering innate immune responses and therefore represents an interesting target for anti-inflammatory therapy.