bluuebunny/crossref_metadata_2025 huggingface dataset:
Critical Role for T Cells in Sephadex-Induced Airway Inflammation: Pharmacological and Immunological Characterization and Molecular Biomarker Identification
Authors: Haddad El-Bdaoui , Underwood Stephen L. , Dabrowski Dominika , Birrell Mark A. , McCluskie Kerryn , Battram Cliff H. , Pecoraro Michaela , Foster Martyn L. , Belvisi Maria G.
Published: April 2014

Intratracheal instillation of Sephadex particles is a convenient model for assessing the impact of potential anti-inflammatory compounds on lung eosinophilia thought to be a key feature in asthma pathophysiology. However, the underlying cellular and molecular mechanisms involved are poorly understood. We have studied the time course of Sephadex-induced lung eosinophilia, changes in pulmonary T cell numbers, and gene and protein expression as well as the immunological and pharmacological modulation of these inflammatory indices in the Sprague Dawley rat. Sephadex increased T cell numbers (including CD4+ T cells) and evoked a pulmonary eosinophilia that was associated with an increase in gene/protein expression of the Th2-type cytokines IL-4, IL-5, and IL-13 and eotaxin in lung tissue. Sephadex instillation also induced airway hyperreactivity to acetylcholine and bradykinin. A neutralizing Ab (R73) against the αβ-TCR caused 54% depletion of total (CD2+) pulmonary T cells accompanied by a significant inhibition of IL-4, IL-13 and eotaxin gene expression together with suppression (65% inhibition) of eosinophils in lung tissue 24 h after Sephadex treatment. Sephadex-induced eosinophilia and Th2 cytokine gene and/or protein expression were sensitive to cyclosporin A and budesonide, compounds that inhibit T cell function, suggesting a pivotal role for T cells in orchestrating Sephadex-induced inflammation in this model.
α-Actinin Is a Cross-Reactive Renal Target for Pathogenic Anti-DNA Antibodies
Authors: Deocharan Bisram , Qing Xiaoping , Lichauco Juan , Putterman Chaim
Published: April 2014

Anti-DNA Abs commonly found in patients with systemic lupus erythematosus are thought to play an important pathogenic role in lupus nephritis. Anti-DNA Abs may contribute to renal disease by cross-reactivity with renal Ags, the identity of which remain elusive. To identify a target Ag for pathogenic anti-DNA Abs, we performed Western blotting and immunoprecipitations of mesangial cell lysates from the lupus-prone MRL-lpr/lpr mouse and a nonautoimmune BALB/c mouse with the pathogenic anti-DNA Ab R4A. We found that R4A (but not a nonpathogenic Ab mutant of R4A) binds to and immunoprecipitates a 100-kDa protein expressed on the cell surface and in lysates of MRL-lpr/lpr mesangial cells. DNase treatment of the lysate and of the R4A Ab did not effect binding, indicating that the binding of R4A to the 100-kDa protein was direct and not mediated by an antigenic bridge containing DNA. Binding was greatly diminished in BALB/c lysates, suggesting that Ag expression or availability at the level of the target organ may be a factor in determining susceptibility to lupus nephritis. Following identification of this 100-kDa protein as nonmuscle α-actinin, binding of R4A to α-actinin was confirmed by Western blot, ELISA, inhibition studies, and immunofluorescence. High titers of anti-α-actinin Abs were present in sera and kidney eluates of lupus mice with active nephritis. These results indicate that the nephritogenicity of some anti-DNA Abs may be mediated via cross-reactivity with α-actinin. Furthermore, variations in target Ag display between individuals may underlie differential susceptibility to anti-DNA Ab-induced renal disease.
A Novel Approach to the Analysis of Specificity, Clonality, and Frequency of HIV-Specific T Cell Responses Reveals a Potential Mechanism for Control of Viral Escape
Authors: Douek Daniel C. , Betts Michael R. , Brenchley Jason M. , Hill Brenna J. , Ambrozak David R. , Ngai Ka-Leung , Karandikar Nitin J. , Casazza Joseph P. , Koup Richard A.
Published: April 2014

Escape from the CD8+ T cell response through epitope mutations can lead to loss of immune control of HIV replication. Theoretically, escape from CD8+ T cell recognition is less likely when multiple TCRs target individual MHC/peptide complexes, thereby increasing the chance that amino acid changes in the epitope could be tolerated. We studied the CD8+ T cell response to six immunodominant epitopes in five HIV-infected subjects using a novel approach combining peptide stimulation, cell surface cytokine capture, flow cytometric sorting, anchored RT-PCR, and real-time quantitative clonotypic TCR tracking. We found marked variability in the number of clonotypes targeting individual epitopes. One subject recognized a single epitope with six clonotypes, most of which were able to recognize and lyse cells expressing a major epitope variant that arose. Additionally, multiple clonotypes remained expanded during the course of infection, irrespective of epitope variant frequency. Thus, CD8+ T cells comprising multiple TCR clonotypes may expand in vivo in response to individual epitopes, and may increase the ability of the response to recognize virus escape mutants.
Cutting Edge: The Minor Histocompatibility Antigen H60 Peptide Interacts with Both H-2Kb and NKG2D
Authors: Cerwenka Adelheid , O’Callaghan Christopher A. , Hamerman Jessica A. , Yadav Rajwardhan , Ajayi Wilfred , Roopenian Derry C. , Joyce Sebastian , Lanier Lewis L.
Published: April 2014

Minor histocompatibility Ags elicit cell-mediated immune responses and graft rejection in individuals receiving MHC-matched tissues. H60 represents a dominant Ag that elicits a strong CTL response in C57BL/6 mice immunized against BALB.B. An 8-aa peptide in the H60 protein is presented by H-2Kb and this is recognized by the TCR as an alloantigen. The intact H60 glycoprotein is a ligand for the costimulatory NKG2D receptor that is expressed by activated CD8+ T cells. Thus, H60 may provide both an allogeneic peptide and its own costimulation. We show that mutation of an H-2Kb-binding anchor residue in the H60 peptide completely abrogates binding of H60 glycoprotein to NKG2D and a synthetic H60 peptide partially blocks the binding of NKG2D to its ligand. Ligands of the human NKG2D receptor are remarkably polymorphic, suggesting that these may also serve as minor histocompatibility Ags.
Cutting Edge: Susceptibility to the Larval Stage of the Helminth ParasiteTaenia crassicepsIs Mediated by Th2 Response Induced Via STAT6 Signaling
Authors: Rodriguez-Sosa Miriam , David John R. , Bojalil Rafael , Satoskar Abhay R. , Terrazas Luis I.
Published: April 2014

Using STAT6−/− BALB/c mice, we analyzed the role of STAT6-induced Th2 response in determining the outcome of murine cysticercosis caused by the helminth parasite Taenia crassiceps. After T. crassiceps infection, wild-type BALB/c mice developed a strong Th2-like response; produced high levels of IgG1, IgE, IL-4, as well as IL-13; and remained susceptible to T. crassiceps. In contrast, similarly infected STAT6−/− mice mounted a strong Th1-like response; produced high levels of IgG2a, IL-12, IFN-γ, as well as nitric oxide; and efficiently controlled T. crassiceps infection. These findings demonstrate that Th2-like response induced via STAT6-mediated signaling pathway mediates susceptibility to T. crassiceps and, furthermore, that unlike the case in most helminths, immunity against T. crassiceps is mediated by a Th1-like rather than Th2-like response.
IFN-γ-Inducible Protein 10 (IP-10; CXCL10)-Deficient Mice Reveal a Role for IP-10 in Effector T Cell Generation and Trafficking
Authors: Dufour Jennifer H. , Dziejman Michelle , Liu Michael T. , Leung Josephine H. , Lane Thomas E. , Luster Andrew D.
Published: April 2014

IFN-γ-inducible protein 10 (IP-10, CXCL10), a chemokine secreted from cells stimulated with type I and II IFNs and LPS, is a chemoattractant for activated T cells. Expression of IP-10 is seen in many Th1-type inflammatory diseases, where it is thought to play an important role in recruiting activated T cells into sites of tissue inflammation. To determine the in vivo function of IP-10, we constructed an IP-10-deficient mouse (IP-10−/−) by targeted gene disruption. Immunological analysis revealed that IP-10−/− mice had impaired T cell responses. T cell proliferation to allogeneic and antigenic stimulation and IFN-γ secretion in response to antigenic challenge were impaired in IP-10−/− mice. In addition, IP-10−/− mice exhibited an impaired contact hypersensitivity response, characterized by decreased ear swelling and reduced inflammatory cell infiltrates. T cells recovered from draining lymph nodes also had a decreased proliferative response to Ag restimulation. Furthermore, IP-10−/− mice infected with a neurotropic mouse hepatitis virus had an impaired ability to control viral replication in the brain. This was associated with decreased recruitment of CD4+ and CD8+ lymphocytes into the brain, reduced levels of IFN-γ and the IFN-γ-induced chemokines monokine induced by IFN-γ (Mig, CXCL9) and IFN-inducible T cell α chemoattractant (I-TAC, CXCL11) in the brain, decreased numbers of virus-specific IFN-γ-secreting CD8+ cells in the spleen, and reduced levels of demyelination in the CNS. Taken together, our data suggest a role for IP-10 in both effector T cell generation and trafficking in vivo.
Secondary Lymphoid Tissue Chemokine (CCL21) Activates CXCR3 to Trigger a Cl− Current and Chemotaxis in Murine Microglia
Authors: Rappert Angelika , Biber Knut , Nolte Christiane , Lipp Martin , Schubel Andreas , Lu Bao , Gerard Norma P. , Gerard Craig , Boddeke Hendrikus W. G. M. , Kettenmann Helmut
Published: April 2014

Microglial cells represent the major immunocompetent element of the CNS and are activated by any type of brain injury or disease. A candidate for signaling neuronal injury to microglial cells is the CC chemokine ligand CCL21, given that damaged neurons express CCL21. Investigating microglia in acute slices and in culture, we demonstrate that a local application of CCL21 for 30 s triggered a Cl− conductance with lasted for tens of minutes. This response was sensitive to the Cl− channel blockers 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and 4-acetamide-4′-isothiocyanatostilbene, 2,2′-disulfonic acid. Moreover, CCL21 triggered a chemotaxis response, which was sensitive to Cl− channel blockers. In microglial cells cultured from CCR7 knockout mice, CCL21 produced the same type of Cl− current as well as a chemotaxis response. In contrast, in microglial cells from CXCR3 knockout mice, CCL21 triggered neither a Cl− conductance nor a chemotaxis response after CCL21 application. We conclude that the CCL21-induced Cl− current is a prerequisite for the chemotaxis response mediated by the activation of CXCR3 but not CCR7 receptors, indicating that in brain CCL21 acts via a different receptor system than in lymphoid organs.
TCR Activation of Human T Cells Induces the Production of Exosomes Bearing the TCR/CD3/ζ Complex
Authors: Blanchard Nicolas , Lankar Danielle , Faure Florence , Regnault Armelle , Dumont Céline , Raposo Graça , Hivroz Claire
Published: April 2014

We show in this study that human T cells purified from peripheral blood, T cell clones, and Jurkat T cells release microvesicles in the culture medium. These microvesicles have a diameter of 50–100 nm, are delimited by a lipidic bilayer membrane, and bear TCR β, CD3ε, and ζ. This microvesicle production is regulated because it is highly increased upon TCR activation, whereas another mitogenic signal, such as PMA and ionomycin, does not induce any release. T cell-derived microvesicles also contain the tetraspan protein CD63, suggesting that they originate from endocytic compartments. They contain adhesion molecules such as CD2 and LFA-1, MHC class I and class II, and the chemokine receptor CXCR4. These transmembrane proteins are selectively sorted in microvesicles because CD28 and CD45, which are highly expressed at the plasma membrane, are not found. The presence of phosphorylated ζ in these microvesicles suggests that the CD3/TCR found in the microvesicles come from the pool of complexes that have been activated. Proteins of the transduction machinery, tyrosine kinases of the Src family, and c-Cbl are also observed in the T cell-derived microvesicles. Our data demonstrate that T lymphocytes produce, upon TCR triggering, vesicles whose morphology and phenotype are reminiscent of vesicles of endocytic origin produced by many cell types and called exosomes. Although the exact content of T cell-derived exosomes remains to be determined, we suggest that the presence of TCR/CD3 at their surface makes them powerful vehicles to specifically deliver signals to cells bearing the right combination of peptide/MHC complexes.
Phenotypic and Functional Differences Between NKT Cells Colonizing Splanchnic and Peripheral Lymph Nodes
Authors: Laloux Véronique , Beaudoin Lucie , Ronet Catherine , Lehuen Agnès
Published: April 2014

NKT cells are considered unconventional T cells. First, they are restricted by a nonclassical MHC class I molecule, CD1d, which presents glycolipids; second, their TCR repertoire is very limited. After stimulation by their TCR, NKT cells rapidly release large amounts of cytokines, such as IL-4 and IFN-γ. Little is known about NKT cells present in lymph nodes. In the present report we show that NKT cells are differently distributed in various lymph nodes and are, for instance, abundant in pancreatic and mesenteric lymph nodes of C57BL/6 mice and nonobese diabetic mice. The high frequency of NKT cells in splanchnic lymph nodes is not simply a consequence of inflammatory signals, as draining lymph nodes still contain low frequencies of NKT cells after IFA or CFA injections. NKT cells from splanchnic lymph nodes harbor a Vβ repertoire similar to that of splenic and liver NKT cells, in contrast to peripheral NKT cells that are not biased toward Vβ8 segments. Analysis of cytokine production by NKT cells from splanchnic lymph nodes reveals that they produce at least as much IL-4 as IFN-γ, in contrast to NKT cells from other organs (spleen, liver, and peripheral lymph nodes), which produce much more IFN-γ than IL-4. These specific features of NKT cells from splanchnic lymph nodes might explain their protective action against the development of pathogenic Th1 cells in type 1 diabetes.
The Cross-Priming APC Requires a Rel-Dependent Signal to Induce CTL
Authors: Mintern Justine D. , Belz Gabrielle , Gerondakis Steve , Carbone Francis R. , Heath William R.
Published: April 2014

Induction of OVA-specific CTL by cross-priming requires help from CD4 T cells, which use CD154 to signal CD40 on the APC. To further dissect the molecular pathways involved in cross-priming, we examined the role of Rel, an NF-κB family member. c-rel−/− mice failed to generate OVA-specific CTL by cross-priming, but could induce CTL to HSV-1. Using chimeric mice, Rel expression was shown to be required by the APC, but not by the T cells. Notably, the deficiency in Rel could be overcome by triggering CD40, implying that the APC required Rel before receipt of the CD40 signal. These data suggest that the cross-priming APC must receive two signals before it can stimulate CTL. The first signal is Rel dependent and is required before activation of CD4 helper T cells, which then deliver the second signal using CD154 to trigger CD40.
Role of TNF Receptor-Associated Factor 2 in the Activation of IgM Secretion by CD40 and CD120b
Authors: Hostager Bruce S. , Bishop Gail A.
Published: April 2014

TNFR-associated factors (TRAFs) participate in the signaling of many TNFR family members, including CD40, CD120a (TNFR1), and CD120b (TNFR2). Previously, we found that a dominant-negative TRAF2 molecule inhibits CD40-mediated Ab secretion by the mouse B cell line CH12.LX. However, disruption of the TRAF2 binding site in the cytoplasmic domain of CD40 does not diminish the ability of CD40 to stimulate Ab secretion, nor is this mutation able to circumvent the inhibition of Ab secretion by dominant-negative TRAF2. Here we demonstrate that CD40-induced TNF stimulates IgM production through CD120b and that CD120b signaling is required for optimal CD40-induced IgM secretion. Furthermore, although both CD40 and CD120b can bind TRAF2, TRAF2-dependent CD40 signals cannot substitute for TRAF2-dependent CD120b signals in the activation of IgM secretion. Our results indicate a potentially important role for CD120b in the activation of IgM secretion and that TRAF2 is used by CD40 and CD120b in distinct ways.
Mutational Analysis of Immunoreceptor Tyrosine-Based Inhibition Motifs of the Ig-Like Transcript 2 (CD85j) Leukocyte Receptor
Authors: Bellón Teresa , Kitzig Friederike , Sayós Joan , López-Botet Miguel
Published: April 2014

The inhibitory receptor Ig-like transcript (ILT)2 (leukocyte Ig-like receptor or CD85j) is a type I transmembrane protein expressed by different leukocyte lineages. The extracellular region of ILT2 binds HLA class I molecules, and its cytoplasmic domain displays four immunoreceptor tyrosine-based inhibition motifs. Upon tyrosine phosphorylation ILT2 recruits the Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) that is involved in negative signaling. To address the structural basis of ILT2-mediated inhibitory signaling, deletion and single tyrosine mutants were generated and transfected in the COS-7 and rat basophilic leukemia cell lines; their abilities to bind SHP-1 and to inhibit FcεR-induced serotonin release in rat basophilic leukemia cells were studied. Both biochemical and functional analyses revealed tyrosines 644 (SIYATL) and 614 (VTYAQL) as the SHP-1 docking sites required for ILT2 inhibitory function. Substitution of tyrosine 562 (VTYAEV) did not alter receptor function. By contrast, mutation of tyrosine 533 (NLYAAV) interfered with ILT2 tyrosine phosphorylation and the subsequent SHP-1 recruitment, thus supporting a regulatory role for this motif.
The MHC Class I Homolog of Human Cytomegalovirus Is Resistant to Down-Regulation Mediated by the Unique Short Region Protein (US)2, US3, US6, and US11 Gene Products
Authors: Park Boyoun , Oh Hokyung , Lee Sungwook , Song Yangsook , Shin Jinwook , Sung Young Chul , Hwang Sue-Yun , Ahn Kwangseog
Published: April 2014

Human CMV encodes four unique short region proteins (US), US2, US3, US6, and US11, each independently sufficient for causing the down-regulation of MHC class I molecules on the cell surface. This down-regulation allows infected cells to evade recognition by cytotoxic T cells but leaves them susceptible to NK cells, which lyse cells that lack class I molecules. Another human CMV-encoded protein, unique long region protein 18 (UL18), is an MHC class I homolog that might provide a mechanism for inhibiting the NK cell response. The sequence similarities between MHC class I molecules and UL18 along with the ability of UL18 to form trimeric complexes with β2-microglobulin and peptides led to the hypothesis that if the US and UL18 gene products coexist temporally during infection, the US proteins might down-regulate UL18 molecules, similar to their action on MHC class I molecules. We show here that temporal expression of US and UL18 genes partially overlaps during infection. However, unlike MHC class I molecules, the MHC class I homolog, UL18, is fully resistant to the down-regulation associated with the US2, US3, US6, and US11 gene products. The specific effect of US proteins on MHC class I molecules, but not on UL18, represents another example of how viral proteins have evolved to evade immune surveillance, avoiding fratricide by specifically targeting host proteins.
Tumor Regression Induced by Intratumor Therapy with a Disabled Infectious Single Cycle (DISC) Herpes Simplex Virus (HSV) Vector, DISC/HSV/Murine Granulocyte-Macrophage Colony-Stimulating Factor, Correlates with Antigen-Specific Adaptive Immunity
Authors: Ali Selman A. , Lynam June , McLean Cornelia S. , Entwisle Claire , Loudon Peter , Rojas José M. , McArdle Stephanie E. B. , Li Geng , Mian Shahid , Rees Robert C.
Published: April 2014

Direct intratumor injection of a disabled infectious single cycle HSV-2 virus encoding the murine GM-CSF gene (DISC/mGM-CSF) into established murine colon carcinoma CT26 tumors induced a significant delay in tumor growth and complete tumor regression in up to 70% of animals. Pre-existing immunity to HSV did not reduce the therapeutic efficacy of DISC/mGM-CSF, and, when administered in combination with syngeneic dendritic cells, further decreased tumor growth and increased the incidence of complete tumor regression. Direct intratumor injection of DISC/mGM-CSF also inhibited the growth of CT26 tumor cells implanted on the contralateral flank or seeded into the lungs following i.v. injection of tumor cells (experimental lung metastasis). Proliferation of splenocytes in response to Con A was impaired in progressor and tumor-bearer, but not regressor, mice. A potent tumor-specific CTL response was generated from splenocytes of all mice with regressing, but not progressing tumors following in vitro peptide stimulation; this response was specific for the gp70 AH-1 peptide SPSYVYHQF and correlated with IFN-γ, but not IL-4 cytokine production. Depletion of CD8+ T cells from regressor splenocytes before in vitro stimulation with the relevant peptide abolished their cytolytic activity, while depletion of CD4+ T cells only partially inhibited CTL generation. Tumor regression induced by DISC/mGM-CSF virus immunotherapy provides a unique model for evaluating the immune mechanism(s) involved in tumor rejection, upon which tumor immunotherapy regimes may be based.
Soluble Fibrinogen Modulates Neutrophil Functionality Through the Activation of an Extracellular Signal-Regulated Kinase-Dependent Pathway
Authors: Rubel Carolina , Fernández Gabriela C. , Rosa Fernanda Alves , Gómez Sonia , Bompadre Macarena Beigier , Coso Omar A. , Isturiz Martín A. , Palermo Marina S.
Published: April 2014

The integrin family not only mediates the recruitment of polymorphonuclear leukocytes (PMN) to sites of inflammation but also regulates several effector functions by binding to specific ligands. We have recently demonstrated that soluble fibrinogen (sFbg) is able to trigger an activating signal in PMN through an integrin-dependent mechanism. This activation results in degranulation, phagocytosis enhancement, and apoptosis delay. The aim of the present work was to further elucidate the molecular events that follow sFbg interaction with CD11b in human PMN, and the participation of this signaling pathway in the regulation of neutrophil functionality. We demonstrate that sFbg triggers a cascade of intracellular signals that lead to focal adhesion kinase and extracellular signal-regulated kinase 1/2 tyrosine phosphorylation. The activation of this mitogen-activated protein kinase pathway plays a central role in the sFbg modulation of secondary granule degranulation, Ab-dependent phagocytosis, and apoptosis. However, fibrinogen-induced secretory vesicle degranulation occurs independently of the signaling transduction pathways investigated herein. In the context of an inflammatory process, the intracellular signal pathway activated by sFbg may be an early event influencing the functionality of PMN.
Contribution of Vascular Endothelial Growth Factor to Airway Hyperresponsiveness and Inflammation in a Murine Model of Toluene Diisocyanate-Induced Asthma
Authors: Lee Yong C. , Kwak Yong-Geun , Song Chang H.
Published: April 2014

Isocyanate chemicals, including toluene diisocyanate (TDI), are currently the most common causes of occupational asthma. Although considerable controversy remains regarding its pathogenesis, TDI-induced asthma is characterized by hyperresponsiveness and inflammation of the airways. One of the histological hallmarks of inflammation is angiogenesis, but the possible role of vascular endothelial growth factor (VEGF), a potent angiogenic cytokine, in TDI-induced asthma is unknown. We developed a murine model to investigate TDI-induced asthma by performing two courses of sensitization with 3% TDI and one challenge with 1% TDI using ultrasonic nebulization to examine the potential involvement of VEGF in that disease. These mice develop the following typical pathophysiological features: airway hyperresponsiveness, airway inflammation, and increased VEGF levels in the airway. Administration of VEGFR inhibitors reduced all these pathophysiological symptoms. These results suggest that VEGF is one of the major determinants of TDI-induced asthma and that the inhibition of VEGF may be a good therapeutic strategy.
Transgenic Expression of a Soluble Complement Inhibitor Protects Against Renal Disease and Promotes Survival in MRL/lprMice
Authors: Bao Lihua , Haas Mark , Boackle Susan A. , Kraus Damian M. , Cunningham Patrick N. , Park Pierce , Alexander Jessy J. , Anderson Randall K. , Culhane Kristin , Holers V. Michael , Quigg Richard J.
Published: April 2014

To investigate the role of complement in lupus nephritis, we used MRL/lpr mice and a transgene overexpressing a soluble complement regulator, soluble CR1-related gene/protein y (sCrry), both systemically and in kidney. Production of sCrry in sera led to significant complement inhibition in Crry-transgenic mice relative to littermate transgene negative controls. This complement inhibition with sCrry conferred a survival advantage to MRL/lpr mice. In a total of 154 animals, 42.5% transgene-negative animals had impaired renal function (blood urea nitrogen > 50 mg/dl) compared with 16.4% mice with the sCrry-producing transgene (p < 0.001). In those animals that died spontaneously, MRL/lpr mice with the sCrry-producing transgene did not die of renal failure, while those without the transgene did (blood urea nitrogen values of 46.6 ± 9 and 122 ± 29 mg/dl in transgene-positive and transgene-negative animals, respectively; p < 0.001). Albuminuria was reduced in those transgenic animals in which sCrry expression was maximally stimulated (urinary albumin/creatinine = 12.4 ± 4.3 and 36.9 ± 7.7 in transgene-positive and transgene-negative animals, respectively; p < 0.001). As expected in the setting of chronic complement inhibition, there was less C3 deposition in glomeruli of sCrry-producing transgenic mice compared with transgene-negative animals. In contrast, there was no effect on glomerular IgG deposition, levels of anti-dsDNA Ab and rheumatoid factor, or spleen weights between the two groups. Thus, long-term complement inhibition reduces renal disease in MRL/lpr mice, which translates into improved survival. MRL/lpr mice in which complement is inhibited still have spontaneous mortality, yet this is not from renal disease.
A Peptide DNA Surrogate Accelerates Autoimmune Manifestations and Nephritis in Lupus-Prone Mice
Authors: Beger Erik , Deocharan Bisram , Edelman Morris , Erblich Bryna , Gu Yun , Putterman Chaim
Published: April 2014

Lupus-associated anti-DNA Abs display features of Ag selection, yet the triggering Ag in the disease is unknown. We previously demonstrated that the peptide DWEYSVWLSN is bound by a pathogenic anti-DNA Ab, and that immunization of nonautoimmune mice with this peptide induces autoantibodies and renal Ig deposition. To elucidate differences in the induced B cell responses in mice genetically predisposed to autoimmunity, young (NZB × NZW)F1 mice were immunized with this peptide DNA mimetope. DWEYSVWLSN-immunized mice had significantly increased IgG anti-dsDNA, anti-laminin, and anti-cardiolipin Ab titers compared with controls. In addition, glomerular histopathology in the form of endocapillary disease and crescent formation was markedly more severe in DWEYSVWLSN-immunized mice. Analysis of mAbs from DWEYSVWLSN-immunized mice revealed that anti-peptide Abs were often cross-reactive with DNA. Genetic elements used in the Ab response in immunized mice were homologous to those used in the spontaneous anti-DNA response in (NZB × NZW)F1 mice, as well as in other, experimentally induced anti-DNA Abs. Our results indicate that peptide immunization can induce a molecular genetic response common to a variety of stimuli that break tolerance to mammalian dsDNA. Based on the similarity between spontaneously arising anti-DNA Abs and several types of induced anti-DNA Abs, we suggest that there may be more than a single Ag that can trigger systemic lupus erythematosus.
NK Cell Receptors of the Orangutan (Pongo pygmaeus): A Pivotal Species for Tracking the Coevolution of Killer Cell Ig-Like Receptors with MHC-C
Authors: Guethlein Lisbeth A. , Flodin Laura R. , Adams Erin J. , Parham Peter
Published: April 2014

CD94, NKG2, Ly49, and killer cell Ig-like receptor (KIR) expressed by orangutan peripheral blood cells were examined by cloning and sequencing cDNA from a panel of individuals. Orthologs of human CD94, NKG2A, D, and F were defined. NKG2C and E are represented by one gene, Popy-NKG2CE, that is equidistant from the two human genes. Several Popy-CD94, NKG2A, and NKG2CE alleles were defined. Popy-Ly49L is expressed in cultured NK cells and has a sequence consistent with it encoding a functional receptor. Orangutan KIR corresponding to the three KIR lineages expressed in humans and chimpanzees were defined. Popy-KIR2DL4 of lineage I is the only ortholog of a human or chimpanzee KIR, but in all individuals examined, the transcripts of this gene produced premature termination, either in the D2 domain or at the beginning of the cytoplasmic domain. Ten Popy-KIR3DL and one Popy-KIR3DS of lineage II are all closely related, but represent the products of at least two genes. The two Popy-KIR2DL and four Popy-KIR2DS of lineage III also represent two genes, both being more related to KIR2DS4 than to other human and chimpanzee KIR of lineage III. The Popy-KIR2D include ones predicted to be specific for the C1 epitope of MHC-C, but none specific for C2. This correlates with the observation that all orangutan MHC-C allotypes examined have the C1 motif.
Genetic Control of Human NK Cell Repertoire
Authors: Shilling Heather G. , Young Neil , Guethlein Lisbeth A. , Cheng Nathalie W. , Gardiner Clair M. , Tyan Dolly , Parham Peter
Published: April 2014

Through differential killer cell Ig-like receptor (KIR) and CD94:NKG2 gene expression, human NK cells generate diverse repertoires, each cell having an inhibitory receptor for autologous HLA class I. Using a new method for measuring repertoire difference that integrates multiple flow cytometry parameters, we found individual repertoire stability, but population variability. Correlating repertoire differences with KIR and HLA genotype for 85 sibling pairs reveals the dominant influence of KIR genotype; HLA genotype having a subtle, modulating effect on relative KIR expression frequencies. HLA and/or KIR genotype also influences CD94:NKG2A expression. After HLA-matched stem cell transplantation, KIR repertoires either recapitulated that of the donor or were generally depressed for KIR expression. Human NK cell repertoires are defined by combinations of variable KIR and HLA class I genes and conserved CD94:NKG2 genes.
Phenotype and Regulation of Persistent Intracerebral T Cells in Murine Toxoplasma Encephalitis
Authors: Schlüter Dirk , Meyer Timothy , Kwok Lai-Yu , Montesinos-Rongen Manuel , Lütjen Sonja , Strack Andreas , Schmitz M. Lienhard , Deckert Martina
Published: April 2014

Toxoplasma gondii is a parasite causing asymptomatic, persistent encephalitis. Protective CD4 and CD8 T cells are recruited to and accumulate in the brain in acute Toxoplasma encephalitis (TE), with slowly decreasing numbers in chronic TE. It is unclear how the size of the intracerebral T cell pool is regulated. Conceivably, permanent recruitment, proliferation, and apoptosis may be involved. We observed that in murine TE recruitment of T cells to the brain was terminated in chronic TE. In vivo 5-bromo-2′-deoxyuridine incorporation and in vitro T cell proliferation experiments revealed that intracerebral T cells did not proliferate, which was explained by the expression of the cell cycle inhibitors p21Waf/cip1 and p27Kip1 and the inhibitory activity of intracerebral F4/80+ cells. TUNEL staining detected apoptotic T cells at low frequency corresponding to an increased expression of the anti-apoptotic molecules Bcl-2 and Bcl-xL and a reduced expression of the pro-apoptotic molecules Bad, Bax, and Fas ligand in CD4 and CD8 T cells. During progression from acute to chronic TE, both CD4 and CD8 T cells down-regulated CD45RB expression and expressed a differential pattern of cytokines. From these experiments it is concluded that the number of intracerebral T cells increases by recruitment of T cells during acute infection, whereas proliferation of intracerebral T cells does not play a role. In chronic TE, T cell recruitment is terminated, the phenotype of intracerebral T cells changes, and their number is gradually downsized by low level apoptosis, which, however, does not completely resolve the T cell infiltrates.
Selective Expression of Type I IFN Genes in Human Dendritic Cells Infected withMycobacterium tuberculosis
Authors: Remoli Maria Elena , Giacomini Elena , Lutfalla Georges , Dondi Elisabetta , Orefici Graziella , Battistini Angela , Uzé Gilles , Pellegrini Sandra , Coccia Eliana M.
Published: April 2014

Type I IFN regulates different aspects of the immune response, inducing a cell-mediated immunity. We have recently shown that the infection of dendritic cells (DC) with Mycobacterium tuberculosis (Mtb) induces IFN-α. In this work we have monitored a rapid induction of IFN-β followed by the delayed production of the IFN-α1 and/or -α13 subtypes. The Mtb infection rapidly activates the NF-κB complex and stimulates the phosphorylation of IFN regulatory factor (IRF)-3, events known to induce IFN-β expression in viral infection. In turn, the autocrine production of IFN-β induces the IFN-stimulated genes that contain binding sites for activated STATs in their promoters. Among the IFN-stimulated genes induced in DC through STAT activation are IRF-1 and IRF-7. The expression of IRF-1 appears to be dependent on the sequential activation of NF-κB and STAT-1. Once expressed, IRF-1 may further stimulate the transcription of IFN-β. Induction of IRF-7 is also regulated at the transcriptional level through the binding of phosphorylated STAT-1 and STAT-2, forming the IFN-stimulated gene factor-3 complex. In turn, the IRF-1 and IRF-7 expression appears to be required for the delayed induction of the IFN-α1/13 genes. Although correlative, our results strongly support the existence of a cascade of molecular events in Mtb-infected DC. Upon infection, constitutively expressed NF-κB and IRF-3 are activated and likely contribute to the rapid IFN-β expression. In turn, IFN-β-induced IRF-1 and IRF-7 may cooperate toward induction of IFN-α1/13 if infection persists and these factors are activated.
Anti-IL-10 Therapeutic Strategy Using the Immunomodulator AS101 in Protecting Mice from Sepsis-Induced Death: Dependence on Timing of Immunomodulating Intervention
Authors: Kalechman Yona , Gafter Uzi , Gal Rivka , Rushkin Galit , Yan Donghong , Albeck Michael , Sredni Benjamin
Published: April 2014

The role of IL-10 in experimental sepsis is controversial. The nontoxic immunomodulator, ammonium trichloro(dioxoethylene-o,o′)tellurate (AS101) has been previously shown to inhibit IL-10 expression at the transcriptional level. In this study, we show that in mice subjected to cecal ligation and puncture (CLP), treatment with AS101 12 h after, but not before, CLP significantly increased survival of septic mice. This was associated with a significant decrease in serum IL-10 and in IL-10 secretion by peritoneal macrophages 24–48 h after CLP. At that time, the ability of these cells to secrete TNF-α and IL-1β was restored in AS101-treated mice. The increased survival of AS101-treated mice was due to the inhibition of IL-10, since cotreatment with murine rIL-10 abolished the protective activity of AS101. AS101 increased class II Ag expression on peritoneal macrophages, severely depressed in control mice, while it did not affect the expression of class I Ags. This was accompanied by a significant elevation in the level of IFN-γ secreted by splenocytes. Moreover, AS101 ameliorated bacterial clearance in the peritoneum and blood and decreased severe multiple organ damage, as indicated by clinical chemistry. Furthermore, myeloperoxidase levels in the liver and lung of AS101-treated mice, an indirect means of determining the recruitment of neutrophils, were significantly decreased. We suggest that nontoxic agents such as AS101, with the capacity to inhibit IL-10 and stimulate macrophage functions, may have clinical potential in the treatment of sepsis, provided they are administered during the phase of sepsis characterized by immune suppression.
Memory Functions and Death Proneness in Three CD4+CD45RO+ Human T Cell Subsets
Authors: Ohara Takaaki , Koyama Kazuaki , Kusunoki Yoichiro , Hayashi Tomonori , Tsuyama Naohiro , Kubo Yoshiko , Kyoizumi Seishi
Published: April 2014

We propose a classification of human CD4+CD45RO+ memory T cells into three new subsets based on cell surface expression levels of CD43. The first subset consists of cells whose CD43 expression is relatively high; this subset also contains the highest proportion of recall Ag-reactive precursors, and its constituent cells respond far more strongly than cells in either of the other subsets to immobilized CD3 Ab in addition to secreting substantially more IFN-γ and IL-4. Cells of the second subset express similar levels of CD43 to naive cells, and they also respond weakly to TCR-mediated stimuli as judged by either their ability to proliferate or capacity for cytokine production. The third subsets consists of cells whose CD43 expression levels are clearly down-regulated; its cells appear to be anergic to TCR-mediated stimuli, and when examined ex vivo many of them appear to be undergoing either spontaneous apoptosis via a caspase-independent pathway or Fas-mediated apoptosis via a caspase-dependent pathway, even in the resting state. An analysis of telomere lengths revealed that the typical telomere of a cell in the second subset was significantly longer than the typical telomere in the first or third subset. Taken together, these results appear to indicate that CD4+CD45RO+ T cells fall into three functionally differing subsets, one being a subset of cells with fully matured memory phenotype, a second being a less mature subset of cells that retain longer telomeres and whose memory functionality is marginal, and a third consisting of anergic cells that give every appearance of being death-prone and/or in the process of dying.
New IL-17 Family Members Promote Th1 or Th2 Responses in the Lung: In Vivo Function of the Novel Cytokine IL-25
Authors: Hurst Stephen D. , Muchamuel Tony , Gorman Daniel M. , Gilbert Jonathan M. , Clifford Theresa , Kwan Sylvia , Menon Satish , Seymour Brian , Jackson Craig , Kung Ted T. , Brieland Joan K. , Zurawski Sandra M. , Chapman Richard W. , Zurawski Gerard , Coffman Robert L.
Published: April 2014

We have biologically characterized two new members of the IL-17 cytokine family: IL-17F and IL-25. In contrast to conventional in vitro screening approaches, we have characterized the activity of these new molecules by direct in vivo analysis and have compared their function to that of other IL-17 family members. Intranasal administration of adenovirus expressing IL-17, IL-17C, or IL-17F resulted in bronchoalveolar lavage neutrophilia and inflammatory gene expression in the lung. In contrast, intranasal administration of IL-25-expressing adenovirus or IL-25 protein resulted in the production of IL-4, IL-5, IL-13, and eotaxin mRNA in the lung and marked eosinophilia in the bronchoalveolar lavage and lung tissue. Mice given intranasal IL-25 also developed epithelial cell hyperplasia, increased mucus secretion, and airway hyperreactivity. IL-25 gene expression was detected following Aspergillus and Nippostrongylus infection in the lung and gut, respectively. IL-25-induced eosinophilia required IL-5 and IL-13, but not IL-4 or T cells. Following IL-25 administration, the IL-5+ staining cells were CD45R/B220+, Thy-1+/−, but were NK1.1-, Ly-6G(GR-1)-, CD4-, CD3-, and c-kit-negative. γ-common knockout mice did not develop eosinophilia in response to IL-25, nor were IL-5+ cells detected. These findings suggest the existence of a previously unrecognized cell population that may initiate Th2-like responses by responding to IL-25 in vivo. Further, these data demonstrate the heterogeneity of function within the IL-17 cytokine family and suggest that IL-25 may be an important mediator of allergic disease via production of IL-4, IL-5, IL-13, and eotaxin.
Complement Deficiency Ameliorates Collagen-Induced Arthritis in Mice
Authors: Hietala Max Albert , Jonsson Ing-Marie , Tarkowski Andrej , Kleinau Sandra , Pekna Marcela
Published: April 2014

Collagen-induced arthritis (CIA) is an experimental animal model of human rheumatoid arthritis being characterized by synovitis and progressive destruction of cartilage and bone. CIA is induced by injection of heterologous or homologous collagen type II in a susceptible murine strain. DBA/1J mice deficient of complement factors C3 (C3−/−) and factor B (FB−/−) were generated to elucidate the role of the complement system in CIA. When immunized with bovine collagen type II emulsified in CFA, control mice developed severe arthritis and high CII-specific IgG Ab titers. In contrast, the C3−/− and FB−/− were highly resistant to CIA and displayed decreased CII-specific IgG Ab response. A repeated bovine collagen type II exposure 3 wk after the initial immunization led to an increase in the Ab response in all mice and triggered arthritis also in the complement-deficient mice. Although the arthritic score of the C3−/− mice was low, the arthritis in FB−/− mice ranked intermediate with regard to C3−/− and control mice. We conclude that complement activation by both the classical and the alternative pathway plays a deleterious role in CIA.
A Role for Oxidative Stress in Apoptosis: Oxidation and Externalization of Phosphatidylserine Is Required for Macrophage Clearance of Cells Undergoing Fas-Mediated Apoptosis
Authors: Kagan Valerian E. , Gleiss Bettina , Tyurina Yulia Y. , Tyurin Vladimir A. , Elenström-Magnusson Carina , Liu Shang-Xi , Serinkan F. Behice , Arroyo Antonio , Chandra Joya , Orrenius Sten , Fadeel Bengt
Published: April 2014

Exposure of phosphatidylserine (PS) on the surface of apoptotic cells has been suggested to serve as an important recognition signal for macrophages. In this work we show that triggering of the death receptor Fas on Jurkat cells results in the generation of reactive oxygen species with oxidation and externalization of PS but not of the other major aminophospholipid, phosphatidylethanolamine. These cells were readily ingested by several classes of macrophages, whereas Raji cells, which are defective for Fas-induced PS exposure, remained unengulfed. However, when Raji cells were incubated with the thiol-reactive agent N-ethylmaleimide to induce PS exposure in the absence of other features of apoptosis, these cells were also engulfed by macrophages. Phagocytosis of Fas-triggered Jurkat cells was inhibited by superoxide dismutase and catalase, which prevent oxidation of PS while allowing PS to remain externalized on these cells. Moreover, liposomes containing oxidized PS (PS-OX) were more potent inhibitors of phagocytosis than those containing its nonoxidized counterpart. Finally, enrichment of the plasma membrane of Jurkat or Raji cells, or myeloid leukemic HL-60 cells, with exogenous PS resulted in phagocytic cell clearance, and this process was further enhanced when PS was substituted for by PS-OX. Taken together, our data suggest that the presence of PS-OX in conjunction with nonoxidized PS on the cell surface is an important signal for macrophage clearance of apoptotic cells.
Generation of CTL Recognizing an HLA-A*0201-Restricted Epitope Shared by MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 Tumor Antigens: Implication in a Broad-Spectrum Tumor Immunotherapy
Authors: Graff-Dubois Stéphanie , Faure Olivier , Gross David-Alexandre , Alves Pedro , Scardino Antonio , Chouaib Salem , Lemonnier François A. , Kosmatopoulos Kostas
Published: April 2014

MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 are expressed in a significant proportion of primary and metastatic tumors of various histological types and are targets of tumor Ag-specific CTL. Individual MAGE-A expression varies from one tumor type to the other but, overall, the large majority of tumors expresses at least one MAGE-A Ag. Therefore, targeting epitopes shared by all MAGE-A Ags would be of interest in immunotherapy against a broad spectrum of cancers. In the present study, we describe a heteroclitic MAGE-A peptide (p248V9) that induces CTL in vivo in HLA-A*0201 transgenic HHD mice and in vitro in healthy donors. These CTL are able to recognize two low HLA-A*0201 affinity peptides differing at their C-terminal position and derived from MAGE-A2, -A3, -A4, -A6, -A10, and -A12 (p248G9) and MAGE-A1 (p248D9). Interestingly, p248V9-specific CTL respond to endogenous MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 in an HLA-A*0201-restricted manner and recognize human HLA-A*0201+MAGE-A+ tumor cells of various histological origin. Therefore, this heteroclitic peptide may be considered as a potent candidate for a broad-spectrum tumor vaccination.
Aberrant Production of IL-12 by Macrophages from Several Autoimmune-Prone Mouse Strains Is Characterized by Intrinsic and Unique Patterns of NF-κB Expression and Binding to the IL-12 p40 Promoter
Authors: Liu Jiajian , Beller David
Published: April 2014

Intrinsic defects in macrophage (Mφ) cytokine production characterize many autoimmune-prone mouse strains. Aberrant levels of IL-12, for example, are produced by Mφ isolated from young mice prone to lupus (MRL and NZB/W) and diabetes (nonobese diabetic (NOD)) well before the appearance of disease signs. Evaluation of the possible mechanism(s) underlying the abnormal regulation of IL-12 in these strains revealed novel patterns of Rel family protein binding to the unique p40 NF-κB site in the IL-12 p40 promoter, whereas binding patterns to Ets and CCAAT enhancer binding protein/β sites were normal. In particular, the heightened production of IL-12 by NOD Mφ is associated with elevated levels of the trans-activating p50/c-Rel (p65) complex compared with the nonfunctional p50/p50 dimer. Conversely, the dramatically impaired production of IL-12 by both NZB/W and MRL/+ Mφ is associated with a predominance of p50/p50 and reduced p50/c-Rel(p65) binding. Mechanistically, the unique pattern seen in the lupus strains reflects elevated p50 and reduced c-Rel nuclear protein levels. In NOD extracts, the level of c-Rel is elevated compared with that in lupus strains, but not when compared with that in normal A/J. However, the extent of c-Rel tyrosine phosphorylation noted in NOD extracts is more than double that seen in any other strain. Levels of p65 were similar in all strains tested. These findings reveal that a common mechanism, involving dysregulation of c-Rel and p50, may be used to determine the aberrant IL-12 levels that have the potential to predispose specific mouse strains to systemic or organ-specific autoimmunity.
IL-12 Administration Leads to a Transient Depletion of T Cells, B Cells, and APCs and Concomitant Abrogation of the HLA-A2.1-Restricted CTL Response in Transgenic Mice
Authors: Peter Katrin , Brunda Michael J. , Corradin Giampietro
Published: April 2014

The injection of a mixture of bona fide T cell epitopes can lead to the occurrence of immunodominance, meaning that the immune response is focused on the recognition of a single epitope or a small portion of the epitopes injected. We have previously demonstrated that the administration of rIL-12 can counteract immunodominance in BALB/c mice. In this study, we show that the administration of rIL-12 to HLA-A2.1 transgenic mice (A2kb mice) abrogates specifically the immune response against HLA-A2.1-restricted HIV epitopes in the spleen. This lack of immune response is most probably due to a transient depletion of B cells, T cells, macrophages, and dendritic cells in this organ. Therefore, our study explains the mechanism of immunosuppression by rIL-12 in vivo.