bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxidant-Mediated Mitochondrial Injury in Eosinophil Apoptosis: Enhancement by Glucocorticoids and Inhibition by Granulocyte-Macrophage Colony-Stimulating Factor
Authors: Gardai Shyra J. , Hoontrakoon Raweewan , Goddard Cally D. , Day Brian J. , Chang Ling Yi , Henson Peter M. , Bratton Donna L.
Published: April 2014

The mainstay of asthma therapy, glucocorticosteroids (GCs) have among their therapeutic effects the inhibition of inflammatory cytokine production and induction of eosinophil apoptosis. In the absence of prosurvival cytokines (e.g., GM-CSF), eosinophils appear to be short-lived, undergoing apoptosis over 96 h in vitro. In a dose-dependent manner, GC further enhances apoptosis, while prosurvival cytokines inhibit apoptosis and antagonize the effect of GC. The mechanisms of eosinophil apoptosis, its enhancement by GC, and antagonism of GC by GM-CSF are not well-understood. As demonstrated in this study, baseline apoptosis of eosinophils resulted from oxidant-mediated mitochondrial injury that was significantly enhanced by GC. Mitochondrial injury was detected by early and progressive loss of mitochondrial membrane potential and the antioxidant protein, Mn superoxide dismutase (SOD). Also observed was the activation/translocation of the proapoptotic protein, Bax, to mitochondria. Underscoring the role of oxidants was the inhibition of mitochondrial changes and apoptosis with culture in hypoxia, or pretreatment with a flavoprotein inhibitor or a SOD mimic. GCs demonstrated early (40 min) and late (16 h) activation of proapoptotic c-Jun NH2-terminal kinase (JNK) and decreased the antiapoptotic protein X-linked inhibitor of apoptosis, a recently demonstrated inhibitor of JNK activation. Similarly, inhibition of JNK prevented GC-enhanced mitochondrial injury and apoptosis. Importantly, GM-CSF prevented GC-induced loss of X-linked inhibitor of apoptosis protein, late activation of JNK, and mitochondrial injury even in the face of unchanged oxidant production, loss of MnSOD, and early JNK activation. These data demonstrate that oxidant-induced mitochondrial injury is pivotal in eosinophil apoptosis, and is enhanced by GC-induced prolonged JNK activation that is in turn inhibited by GM-CSF.
CCR8 Is Not Essential for the Development of Inflammation in a Mouse Model of Allergic Airway Disease
Authors: Chung Chan D. , Kuo Frederick , Kumer Jeffrey , Motani Alykhan S. , Lawrence Christopher E. , Henderson William R. , Venkataraman Chandrasekar
Published: April 2014

Chemokine receptors play an important role in the trafficking of various immune cell types to sites of inflammation. Several chemokine receptors are differentially expressed in Th1 and Th2 effector populations. Th2 cells selectively express CCR3, CCR4, and CCR8, which could direct their trafficking to sites of allergic inflammation. Additionally, increased expression of the CCR8 ligand, TCA-3, has been detected in affected lungs in a mouse model of asthma. In this study, CCR8-deficient mice were generated to address the biological role of CCR8 in a model of allergic airway disease. Using two different protocols of allergen challenge, we demonstrate that absence of CCR8 does not affect the development of pulmonary eosinophilia and Th2 cytokine responses. In addition, administration of anti-TCA-3-neutralizing Ab during allergen sensitization and rechallenge failed to inhibit airway allergic inflammation. These results suggest that CCR8 does not play an essential role in the pathogenesis of inflammation in this mouse model of allergic airway disease.
Antagonist of Secondary Lymphoid-Tissue Chemokine (CCR Ligand 21) Prevents the Development of Chronic Graft-Versus-Host Disease in Mice
Authors: Sasaki Miho , Hasegawa Hitoshi , Kohno Masashi , Inoue Atsushi , Ito Mitsuko R. , Fujita Shigeru
Published: April 2014

The use of receptor antagonists for chemokines is an alternative approach to blocking chemokine actions and has the potential to provide novel therapeutics. We determined the receptor antagonist properties of murine N-terminally truncated secondary lymphoid tissue chemokine (SLC)/6Ckine/CCR ligand 21 analogs and evaluated the preventive effects of SLC antagonists on chronic graft-vs-host disease (GVHD) in a murine model by blocking the homing of donor CCR7-expressing T cells into the recipient’s lymphoid organs. SLC analogs truncated >4 aa residues from the N terminus showed a loss of chemotaxis and Ca2+ influx of CCR7-expressing cells and also inhibited SLC-stimulated chemotaxis and SLC-induced Ca2+ influx completely. To determine whether SLC antagonist inhibits the development of chronic GVHD, chronic GVHD was induced by injecting DBA/2 spleen cells into (C57BL/6 × DBA/2) F1 mice. Total numbers of spleen cells and host B cells, serum levels of IgE, and of total IgG and IgG1 of anti-DNA Abs in SLC antagonist-treated GVHD mice were significantly lower than those in control PBS-treated GVHD mice. This was due to a reduction in the levels of activated donor CD4+ T cells and a decrease in IL-4 production, resulting in a reduction in the numbers of activated host B cells. Therefore, our results suggest that SLC antagonist has beneficial effects for the prevention of chronic GVHD.
Impaired Accumulation and Function of Memory CD4 T Cells in Human IL-12 Receptor β1 Deficiency
Authors: Cleary Aileen M. , Tu Wenwei , Enright Andrea , Giffon Thierry , Dewaal-Malefyt Rene , Gutierrez Kathleen , Lewis David B.
Published: April 2014

Defects in IL-12 production or IL-12 responsiveness result in a vulnerability to infection with non-viral intracellular organisms, but the immunological mechanisms responsible for this susceptibility remain poorly understood. We present an immunological analysis of a patient with disseminated Salmonella enteritidis and a homozygous splice acceptor mutation in the IL-12Rβ1-chain gene. This mutation resulted in the absence of IL-12Rβ1 protein on PBMC and an inability of T cells to specifically bind IL-12 or produce IFN-γ in response to either IL-12 or IL-23. The accumulation of memory (CD45R0high) CD4 T cells that were CCR7high (putative central memory cells) was normal or increased for age. Central memory CD4 T cells of the patient and age-matched controls were similar in having a low to undetectable capacity to produce IFN-γ after polyclonal stimulation. In contrast, the patient had a substantial decrease in the number of CCR7neg/dull CD45R0high memory CD4 T cells (putative effector memory cells), and these differed from control cells in having a minimal ability to produce IFN-γ after polyclonal stimulation. Importantly, tetanus toxoid-specific IFN-γ production by PBMC from the patient was also significantly reduced compared with that in age-matched controls, indicating that signaling via the IL-12Rβ1-chain is generally necessary for the in vivo accumulation of human memory CD4 T cells with Th1 function. These results are also consistent with a model in which the IL-12Rβ1 subunit is necessary for the conversion of central memory CD4 T cells into effector memory cells.
Autoimmune Encephalomyelitis and Uveitis Induced by T Cell Immunity to Self β-Synuclein
Authors: Mor Felix , Quintana Francisco , Mimran Avishai , Cohen Irun R.
Published: April 2014

β-synuclein is a neuronal protein that accumulates in the plaques that characterize neurodegenerative diseases such as Parkinson’s and Alzheimer’s diseases. It has been proposed that immunization to peptides of plaque-forming proteins might be used therapeutically to help dissociate pathogenic plaques in the brain. We now report that immunization of Lewis rats with a peptide from β-synuclein resulted in acute paralytic encephalomyelitis and uveitis. T cell lines and clones reactive to the peptide adoptively transferred the disease to naive rats. Immunoblotting revealed the presence of β-synuclein in heavy myelin, indicating that the expression of β-synuclein is not confined to neurons. These results add β-synuclein to the roster of encephalitogenic self Ags, point out the potential danger of therapeutic autoimmunization to β-synuclein, and alert us to the unsuspected possibility that autoimmunity to β-synuclein might play an inflammatory role in the pathogenesis of neurodegeneration.
Dichotomy of Autoreactive Th1 and Th2 Cell Responses to Desmoglein 3 in Patients with Pemphigus Vulgaris (PV) and Healthy Carriers of PV-Associated HLA Class II Alleles
Authors: Veldman Christian , Stauber Angelika , Wassmuth Ralf , Uter Wolfgang , Schuler Gerold , Hertl Michael
Published: April 2014

Pemphigus vulgaris (PV) is the most severe autoimmune bullous skin disorder and is primarily associated with circulating autoantibodies (autoAb) against desmoglein 3 (Dsg3). In light of recent evidence that autoreactive T cells are critical for the induction and regulation of Ab production, the goal of this study was to characterize and quantitate autoreactive T cells in patients with PV and healthy controls. Peripheral Dsg3-reactive Th cells from 28 patients with acute-onset, chronic active, and remittent PV were quantitated by MACS secretion assay. Dsg3-reactive Th2 cells were detected at similar frequencies in all studied PV patients, while the number of autoreactive Th1 cells exceeded those of Th2 cells in chronic active PV. In contrast, healthy carriers of the PV-associated HLA class II alleles, DRB1*0402 and DQB1*0503, exhibited exclusively Dsg3-reactive Th1 cell responses, while healthy carriers of other HLA class II alleles did not. Moreover, the presence of IgG1 and IgG4 against Dsg3 was directly related to the ratio of Dsg3-reactive Th1/Th2 cells. T cell recognition of Dsg3 was restricted by HLA-DRB1*0402 and DQB1*0503 in PV patients and Dsg3-responsive healthy donors. These observations strongly suggest 1) that the appearance of Dsg3-reactive Th2 cells is restricted to patients with PV; 2) that specific HLA class II alleles that are prevalent in PV are critical for T cell recognition of Dsg3 in PV patients and Dsg3-responsive healthy donors; and 3) that autoAb production is associated with both Th1 and Th2 cells.
OX40 Ligand-Transduced Tumor Cell Vaccine Synergizes with GM-CSF and Requires CD40-Apc Signaling to Boost the Host T Cell Antitumor Response
Authors: Gri Giorgia , Gallo Elena , Di Carlo Emma , Musiani Piero , Colombo Mario P.
Published: April 2014

Efficient T cell priming by GM-CSF and CD40 ligand double-transduced C26 murine colon carcinoma is not sufficient to cure metastases in a therapeutic setting. To determine whether a cellular vaccine that interacts directly with both APC and T cells in vivo might be superior, we generated C26 carcinoma cells transduced with the T cell costimulatory molecule OX40 ligand (OX40L) either alone (C26/OX40L) or together with GM-CSF (C26/GM/OX40L), which is known to activate APC. Mice injected with C26/OX40L cells displayed only a delay in tumor growth, while the C26/GM/OX40L tumor regressed in 85% of mice. Tumor rejection required granulocytes, CD4+, CD8+ T cells, and APC-mediated CD40-CD40 ligand cosignaling, but not IFN-γ or IL-12 as shown using subset-depleted and knockout (KO) mice. CD40KO mice primed with C26/GM/OX40L cells failed to mount a CTL response, and T cells infiltrating the C26/GM/OX40L tumor were OX40 negative, suggesting an impairment in APC-T cell cross-talk in CD40KO mice. Indeed, CD4+ T cell-depleted mice failed to mount any CTL activity against the C26 tumor, while treatment with agonistic mAb to CD40, which acts on APC, bypassed the requirement for CD4+ T cells and restored CTL activation. C26/GM/OX40L cells cured 83% of mice bearing lung metastases, whereas C26/OX40L or C26/GM vaccination cured only 28 and 16% of mice, respectively. These results indicate the synergistic activity of OX40L and GM-CSF in a therapeutic setting.
Cutting Edge: IFN Consensus Sequence Binding Protein/IFN Regulatory Factor 8 Drives the Development of Type I IFN-Producing Plasmacytoid Dendritic Cells
Authors: Tsujimura Hideki , Tamura Tomohiko , Ozato Keiko
Published: April 2014

IFN consensus sequence binding protein (ICSBP/IFN regulatory factor 8) is a hematopoietic cell-specific transcription factor essential for the generation of CD8α+ dendritic cells (DCs). We found that ICSBP−/− mice lack B220+CD11b− plasmacytoid DCs (pDCs) in addition to CD8α+ DCs. Although ICSBP−/− mice have B220−CD11b+ myeloid DCs (mDCs), they fail to mature upon Toll-like receptor signaling. Accordingly, ICSBP−/− bone marrow progenitor cells were Tefective in generating pDCs in the fms-like tyrosine kinase 3 ligand-based culture system and mDCs generated in this system were defective in maturation. We demonstrate that introduction of ICSBP rescues the development of pDCs from −/− bone marrow progenitors. ICSBP also restored the ability of both pDCs and mDCs to mature after Toll-like receptor signals. ICSBP-restored DCs produced IFN-α and IL-12p40 in a DC subset-selective manner with the amounts comparable to those by +/+ DCs. Together, ICSBP is essential for early pDC development and final maturation of both pDCs and mDCs.
Cutting Edge: Profile of Chemokine Receptor Expression on Human Plasma Cells Accounts for Their Efficient Recruitment to Target Tissues
Authors: Nakayama Takashi , Hieshima Kunio , Izawa Dai , Tatsumi Youichi , Kanamaru Akihisa , Yoshie Osamu
Published: April 2014

We systematically examined the repertoire of chemokine receptors expressed by human plasma cells. Fresh bone marrow plasma cells and myeloma cells consistently expressed CXCR4, CXCR6, CCR10, and CCR3. Accordingly, plasma cells responded to their respective ligands in chemotaxis and very late Ag-4-dependent cell adhesion to fibronectin. Immobilized CXC chemokine ligand (CXCL)16, a novel transmembrane-type chemokine and CXCR6 ligand, also directly induced adhesion of plasma cells without requiring Gαi signaling or divalent cations. Furthermore, we revealed consistent expression of CXCL12 (CXCR4 ligand), CXCL16 (CXCR6 ligand), and CC chemokine ligand 28 (CCR10 and CCR3 ligand) in tissues enriched with plasma cells including bone marrow, and constitutive expression of CXCL12, CXCL16, and CC chemokine ligand 28 by cultured human bone marrow stromal cells. Collectively, plasma cells are likely to be recruited to bone marrow and other target tissues via CXCR4, CXCR6, CCR10, and CCR3. CXCR6 may also contribute to tissue localization of plasma cells through its direct binding to membrane-anchored CXCL16.
IFN-αβ Released by Mycobacterium tuberculosis-Infected Human Dendritic Cells Induces the Expression of CXCL10: Selective Recruitment of NK and Activated T Cells
Authors: Lande Roberto , Giacomini Elena , Grassi Tiziana , Remoli Maria Elena , Iona Elisabetta , Miettinen Minja , Julkunen Ilkka , Coccia Eliana M.
Published: April 2014

We recently reported that dendritic cells (DC) infected with Mycobacterium tuberculosis (Mtb) produce Th1/IFN-γ-inducing cytokines, IFN-αβ and IL-12. In the present article, we show that maturing Mtb-infected DC express high levels of CCR7 and they become responsive to its ligand CCL21. Conversely, CCR5 expression was rapidly lost from the cell surface following Mtb infection. High levels of CCL3 and CCL4 were produced within 8 h after infection, which is likely to account for the observed CCR5 down-modulation on Mtb-infected DC. In addition, Mtb infection stimulated the secretion of CXCL9 and CXCL10. Interestingly, the synthesis of CXCL10 was mainly dependent on the Mtb-induced production of IFN-αβ. Indeed, IFN-αβ neutralization down-regulated CXCL10 expression, whereas the expression of CXCL9 appeared to be unaffected. The chemotactic activity of the Mtb-infected DC supernatants was evaluated by migration assays using activated NK, CD4+, and CD8+ cells that expressed both CCR5 and CXCR3. Mtb-induced expression of CCL3, CCL4, CXCL9, and CXCL10 was involved in the stimulation of NK and T cell migration. In accordance with the data on the IFN-αβ-induced expression of CXCL10, neutralization of IFN-αβ significantly reduced the chemotactic activity of the supernatant from Mtb-infected DC. This indicates that IFN-αβ may modulate the immune response through the expression of CXCL10, which along with CXCL9, CCL3, and CCL4 participates in the recruitment and selective homing of activated/effector cells, which are known to accumulate at the site of Mtb infection and take part in the formation of the granulomas.
Tumor Eradication by Hepatitis B Virus X Antigen-Specific CD8+ T Cells in Xenografted Nude Mice
Authors: Chun Eunyoung , Lee Jihyun , Cheong Hong Seok , Lee Ki-Young
Published: April 2014

We have previously reported several CTL epitopes derived from the hepatitis B viral X Ag (HBx). In this study, we evaluated whether HBx-specific CTLs can be effectively used in adoptive cancer immunotherapy. To validate the possibility, four peptides containing a HLA-A2.1-restricted binding consensus motif were identified from the HBx protein and tested for their ability to activate CTL from PBMCs isolated from chronic carriers of HBV (n = 12). We selected two highly potent epitopes, HBx 52–60 (HLSLRGLFV) and HBx 115–123 (CLFKDWEEL), that are capable of inducing Ag-specific cytotoxic T cells in patient PBMCs. For adoptive immunotherapy using HBx-specific CTLs, we generated CTL clones restricted to the HBx 52–60 or HBx 115–123 peptide using a limiting dilution technique. LC-46, an HBx 52–60-specific clone, is CD62L−CD69+CD45RO+CD45RA−CD25dim and is stained by IFN-γ (∼92%), IL-2 (30%), and TNF-α (56%), but not by IL-5, IL-10, IL-12, or TNF-β, indicating that the cells are fully activated T cytotoxic 1-type cells. When LC-46 cells were adoptively transferred into xenografted nude mice bearing human hepatomas expressing HLA-A2.1 molecules and intracellular HBx proteins, the tumors were eradicated. Taken together, our data provide solid evidence for the feasibility of adoptive immunotherapy with HBx-sensitized CTLs in hepatitis disease, including hepatocellular carcinoma (HCC).
Cross-Presentation of HLA Class I Epitopes from Exogenous NY-ESO-1 Polypeptides by Nonprofessional APCs
Authors: Gnjatic Sacha , Atanackovic Djordje , Matsuo Mitsutoshi , Jäger Elke , Lee Sang Yull , Valmori Danila , Chen Yao-Tseng , Ritter Gerd , Knuth Alexander , Old Lloyd J.
Published: April 2014

NY-ESO-1, a germ cell Ag often detected in tumor tissues, frequently elicits Ab and CD8+ T cell responses in cancer patients. Overlapping long peptides spanning the NY-ESO-1 sequence have been used to map HLA class I-restricted epitopes recognized by NY-ESO-1-specific CD8+ T lymphocytes. To address the antigenicity of long peptides, we analyzed two synthetic 30-mer peptides from NY-ESO-1, polypeptides 80–109 and 145–174, for their capacity to be processed by APCs and to stimulate CD8+ T cells. By incubating APCs with polypeptides at different temperatures or in the presence of protease inhibitors, we found that NY-ESO-1 polypeptides were rapidly internalized by B cells, T2 cells, or PBLs and submitted to cellular proteolytic action to yield nonamer epitopes presented by HLA class I. Polypeptides were also immunogenic in vitro and stimulated the expansion of CD8+ T cells against naturally processed NY-ESO-1 epitopes in the context of three different HLA class I alleles. Polypeptides can thus serve as exogenous Ags that are cross-presented on HLA class I without requiring the action of professional APCs. These findings support innovative vaccination strategies using NY-ESO-1 polypeptides that would circumvent current limitations of HLA class I peptide vaccination, i.e., HLA eligibility criteria and knowledge of epitope, while allowing for facilitated immunogenicity in the presence of helper epitopes.
The Contribution of NKT Cells, NK Cells, and Other γ-Chain-Dependent Non-T Non-B Cells to IL-12-Mediated Rejection of Tumors
Authors: Park Se-Ho , Kyin Tim , Bendelac Albert , Carnaud Claude
Published: April 2014

IL-12 is a potent cytokine that impairs the growth of several tumors in vivo in natural as well as in therapeutic conditions. Although IL-12 can enhance a number of immunological antitumor mechanisms, including those mediated by NK cells and CTL, recent reports have suggested that the mouse CD1d-restricted Vα14-Jα18 NKT cell was the essential cell type recruited in most, if not all tumor rejection models, including the B16 melanoma. In this study, we have examined and compared the role of NKT cells, T cells, NK cells, and other non-T non-B cells in the rejection of B16 melanoma cells after exogenous administration of IL-12. Surprisingly, our results failed to confirm a necessary role for NKT cells in this model. Instead, we found that NK cells mediated the rejection of liver metastases, whereas other γc-dependent non-T non-B cells, possibly lymphoid dendritic cells, were required for rejection of skin tumors. These findings challenge the view that NKT cells are systematically required for IL-12-mediated rejection of tumors, and instead reveal that a variety of effector pathways can be recruited depending on the tumor microenvironment.
Broadening of Epitope Recognition During Immune Rejection of ErbB-2-Positive Tumor Prevents Growth of ErbB-2-Negative Tumor
Authors: Pilon Shari A. , Kelly Carmen , Wei Wei-Zen
Published: April 2014

Tumor heterogeneity is a limiting factor in Ag-specific vaccination. Ag-negative variants may arise after tumor cells bearing the immunizing Ags are destroyed. In situ priming to tumor-associated epitopes distinct from and not cross-reactive with the immunizing Ags may be crucial to the ultimate success of cancer vaccination. Immunization of BALB/c mice with DNA encoding wild-type human ErbB-2 (Her-2/neu, E2) or cytoplasmic ErbB-2 (cytE2), activated primarily CD4 or CD8 T cells, respectively, and both vaccines protected against ErbB-2-positive D2F2/E2 tumors. In ≥50% of protected mice, a second challenge of ErbB-2-negative D2F2 tumor cells was rejected. Recognition of non-ErbB-2, tumor-associated Ags was demonstrated by immune cell proliferation upon stimulation with irradiated D2F2 cells. This broadening of epitope recognition was abolished if CD4 T cells were depleted before D2F2/E2 tumor challenge, demonstrating their critical role in Ag priming. Similarly, mice that rejected D2F2/cytE2 tumor cells, which express only MHC I epitopes of ErbB-2, were not protected from a second challenge with D2F2 cells. Depletion of CD8 T cells abolished protection against D2F2, indicating the activation of D2F2-specific CTL. Therefore, long term protection may be achieved by immunization with dominant Ag(s), followed by a general enhancement of CD4 T cell activity to promote priming to multiple tumor-associated Ags.
Transfer of TCR Genes into Mature T Cells Is Accompanied by the Maintenance of Parental T Cell Avidity
Authors: Rubinstein Mark P. , Kadima Andre N. , Salem Mohamed L. , Nguyen Christophe L. , Gillanders William E. , Nishimura Michael I. , Cole David J.
Published: April 2014

The adoptive transfer of tumor-specific T cells expanded in vitro can be of significant therapeutic value in select cancer patients. This strategy is limited though, as it is often difficult, if not impossible, to obtain T cells of clinical value. The transfer of TCR genes to mature T cells to generate tumor-reactive T cells provides a potential mechanism to overcome these limitations. To evaluate the feasibility of such an approach and the quality of the resulting T cells, we generated replication-deficient retroviral vectors using the well-characterized OT-1 TCR genes. After transducing murine T cells, we were able to expand large numbers of Ag-specific T cells that were functionally active against tumor cells expressing the relevant Ag. Furthermore, we found that T cells expressing retrovirally encoded TCR had avidity that was similar to that of the parental clone. This maintenance of avidity was despite variable expression of the retrovirally encoded TCR and the presence of potentially competing endogenous TCRs. These results suggest that the inherent qualities of the TCR, as dictated by the coding sequence, are the most critical parameters in the generation of high-avidity T cells.
Differential Antigen Sensitivity and Costimulatory Requirements in Human Th1 and Th2 Antigen-Specific CD4+ Cells with Similar TCR Avidity
Authors: Holzer Ursula , Kwok William W. , Nepom Gerald T. , Buckner Jane Hoyt
Published: April 2014

The differentiation of naive CD4+ Th cells into Th1 and Th2 phenotypes is influenced by cytokines, concentration of Ag, accessory molecules, and the affinity of the MHC-TCR interaction. To study these factors in human memory T cells, T cell lines with Th1 or Th2 phenotypes specific for the peptide hemagglutinin (HA)307–319 in the context of DRB1*0401 were established from the peripheral blood of an individual previously vaccinated for influenza virus. Flow cytometric analysis with fluorescent-labeled MHC class II tetramers was used to analyze TCR avidity: the Th2 line bound the HLA-DR*0401-HA307–319 tetramers with higher mean avidity, although the range of binding avidity largely overlapped with the Th1 line. High-affinity Th1 and Th2 lines were established for further study by FACS sorting. When activated with plate-bound HLA-DR*0401-HA307–319 monomers, the Th1 line proliferated and produced IFN-γ without additional costimulation whereas the Th2 line required the addition of soluble anti-CD28 Ab to induce proliferation and IL-5 production, but this requirement could be overcome with high concentrations of plate-bound monomer alone. IL-2 production was dependent on costimulation in both cell lines. These findings demonstrate that upon antigenic rechallenge, Th1 and Th2 cells differ in their response to Ag-specific stimulation. Th2 cells were sensitive to the strength of signal to a greater degree than Th1 cells and required costimulation through CD28 for maximal proliferation. These distinctions between Th1 and Th2 activation are not consistent with a simple avidity model of Ag recognition and indicate both qualitative and quantitative differences in determining cell lineage commitment.
Critical Role of MHC Class I-Related Chain A and B Expression on IFN-α-Stimulated Dendritic Cells in NK Cell Activation: Impairment in Chronic Hepatitis C Virus Infection
Authors: Jinushi Masahisa , Takehara Tetsuo , Kanto Tatsuya , Tatsumi Tomohide , Groh Veronika , Spies Thomas , Miyagi Takuya , Suzuki Takahiro , Sasaki Yutaka , Hayashi Norio
Published: April 2014

Dendritic cells (DCs) augment effector functions of NK cells, but the underlying mechanisms are not fully understood. Here we show in an in vitro coculture system that human monocyte-derived DCs enhance IFN-γ production, CD69 expression, and K562 cytolytic ability of NK cells when DCs are prestimulated with various maturation stimuli such as IFN-α or LPS. Of interest is the finding that NK cell activation mediated by LPS-stimulated DCs was dependent on IL-12 produced in DC/NK coculture, but that IFN-α-stimulated DC-mediated activation was not. Alternatively, MHC class I-related chain A and B (MICA/B), ligands for NKG2D activating receptor, were found to be induced on DCs upon IFN-α stimulation and to be responsible for the NK activation because mAb-mediated masking of MICA/B as well as inhibition of direct cell-to-cell contact using transwell insert completely abolished DC-dependent NK cell activation by IFN-α. Finally, DCs recovered from chronic hepatitis C virus-infected patients showed defects in the induction of MICA/B and impaired ability to activate NK cells in response to IFN-α stimulation. These findings suggested that MICA/B induction on DCs may be one of the mechanisms by which IFN-α activates NK cells; this impairment might affect IFN-α responsiveness in hepatitis C virus infection.
Blockade of Programmed Death-1 Ligands on Dendritic Cells Enhances T Cell Activation and Cytokine Production
Authors: Brown Julia A. , Dorfman David M. , Ma Feng-Rong , Sullivan Elizabeth L. , Munoz Oliver , Wood Clive R. , Greenfield Edward A. , Freeman Gordon J.
Published: April 2014

Programmed death-1 ligand (PD-L)1 and PD-L2 are ligands for programmed death-1 (PD-1), a member of the CD28/CTLA4 family expressed on activated lymphoid cells. PD-1 contains an immunoreceptor tyrosine-based inhibitory motif and mice deficient in PD-1 develop autoimmune disorders suggesting a defect in peripheral tolerance. Human PD-L1 and PD-L2 are expressed on immature dendritic cells (iDC) and mature dendritic cells (mDC), IFN-γ-treated monocytes, and follicular dendritic cells. Using mAbs, we show that blockade of PD-L2 on dendritic cells results in enhanced T cell proliferation and cytokine production, including that of IFN-γ and IL-10, while blockade of PD-L1 results in similar, more modest, effects. Blockade of both PD-L1 and PD-L2 showed an additive effect. Both whole mAb and Fab enhanced T cell activation, showing that PD-L1 and PD-L2 function to inhibit T cell activation. Enhancement of T cell activation was most pronounced with weak APC, such as iDCs and IL-10-pretreated mDCs, and less pronounced with strong APC such as mDCs. These data are consistent with the hypothesis that iDC have a balance of stimulatory vs inhibitory molecules that favors inhibition, and indicate that PD-L1 and PD-L2 contribute to the poor stimulatory capacity of iDC. PD-L1 expression differs from PD-L2 in that PD-L1 is expressed on activated T cells, placental trophoblasts, myocardial endothelium, and cortical thymic epithelial cells. In contrast, PD-L2 is expressed on placental endothelium and medullary thymic epithelial cells. PD-L1 is also highly expressed on most carcinomas but minimally expressed on adjacent normal tissue suggesting a role in attenuating antitumor immune responses.
Enhanced Differentiation of Splenic Plasma Cells but Diminished Long-Lived High-Affinity Bone Marrow Plasma Cells in Aged Mice
Authors: Han Shuhua , Yang Kaiyong , Ozen Zeynep , Peng Weiyi , Marinova Ekaterina , Kelsoe Garnett , Zheng Biao
Published: April 2014

In the present work, we have dissected the mechanisms responsible for the impaired humoral responses in aging. We found that there was a substantially higher level of Ab-forming cells in the spleens of aged mice than that of young controls. However, the number of high-affinity, class-switched Ab-forming cells was severely decreased in the spleen of aged mice. The accumulation of low-affinity IgM Ab-forming cells in the spleens of aged animals was not due to a deficiency in isotype switching because the number of total IgG1 splenic plasma cells was not significantly reduced. Remarkably, plasma cells of both low and high affinity were significantly diminished in the bone marrow of aged mice compared with that of young mice. The results from reconstitution experiments showed that aged bone marrow was less supportive for plasma cells derived from young splenic B cells. These findings suggest that humoral immune deficiency in aging results from at least two mechanisms: the inability to generate sufficient numbers of high-affinity Ab-forming cells, which is a result of diminished germinal center reaction, and the defective bone marrow environment that has diminished ability to support the selection and survival of long-term Ab-forming cells.
Ly6C Induces Clustering of LFA-1 (CD11a/CD18) and Is Involved in Subtype-Specific Adhesion of CD8 T Cells
Authors: Jaakkola Ilkka , Merinen Marika , Jalkanen Sirpa , Hänninen Arno
Published: April 2014

Ly6C is a hemopoietic cell differentiation Ag found on a subset of CD8 T cells in the periphery. It is involved in target cell killing by CTLs, augments TCR-mediated activation of IL-2 and IFN-γ production in CD8 T cells, and regulates CD8 T cell homing in vivo. In this study, we show that cross-linking of Ly6C causes clustering of LFA-1 (CD11a/CD18) on the surface of CD8 T cells via a mechanism dependent on reorganization of actin cytoskeleton and intracellular protease, calpain, but not the phosphatidylinositol 3-kinase pathway. In the capillary flow-adhesion assay, Ly6C cross-linking significantly augments lymphocyte adhesion to endothelium, and this is inhibited by an Ab that blocks LFA-1 function. Furthermore, upon in vitro cross-linking and during in vivo homing into lymph nodes, Ly6C is transiently lost from cell surface but becomes re-expressed on lymph node-resident CD8 T cells. The abilities of Ly6C to induce LFA-1 clustering and to be re-expressed after signaling-associated down-regulation may be important in regulating the homing of CD8 T cells into lymph nodes and in subsequent steps of CD8 T cell activation and effector function that again involve LFA-1.
TGF-β Regulates Airway Responses Via T Cells
Authors: Schramm Christoph , Herz Udo , Podlech Jürgen , Protschka Martina , Finotto Susetta , Reddehase Matthias J. , Köhler Heinz , Galle Peter R. , Lohse Ansgar W. , Blessing Manfred
Published: April 2014

Allergic asthma is characterized by airway hyperreactivity, inflammation, and a Th2-type cytokine profile favoring IgE production. Beneficial effects of TGF-β and conflicting results regarding the role of Th1 cytokines have been reported from murine asthma models. In this study, we examined the T cell as a target cell of TGF-β-mediated immune regulation in a mouse model of asthma. We demonstrate that impairment of TGF-β signaling in T cells of transgenic mice expressing a dominant-negative TGF-β type II receptor leads to a decrease in airway reactivity in a non-Ag-dependent model. Increased serum levels of IFN-γ can be detected in these animals. In contrast, after injection of OVA adsorbed to alum and challenge with OVA aerosol, transgenic animals show an increased airway reactivity and inflammation compared with those of wild-type animals. IL-13 levels in bronchoalveolar lavage fluid and serum as well as the number of inducible NO synthase-expressing cells in lung infiltrates were increased in transgenic animals. These results demonstrate an important role for TGF-β signaling in T cells in the regulation of airway responses and suggest that the beneficial effects observed for TGF-β in airway hyperreactivity and inflammation may be due to its regulatory effects on T cells.
TNF-α Plus IFN-γ Induce Connexin43 Expression and Formation of Gap Junctions Between Human Monocytes/Macrophages That Enhance Physiological Responses
Authors: Eugenín Eliseo A. , Brañes María C. , Berman Joan W. , Sáez Juan C.
Published: April 2014

In this work, the effects of bacterial LPS, TNF-α, and IFN-γ on gap junctional communication (dye coupling) and on the expression of connexin43 (immunofluorescence, immunoblotting, and RT-PCR) in monocytes/macrophages were studied. Freshly isolated human monocytes plated at high density and treated either with LPS plus IFN-γ or TNF-α plus IFN-γ became transiently dye coupled (Lucifer yellow) within 24 h. Cells treated with LPS, TNF-α, or IFN-γ alone remained dye uncoupled. In dye-coupled cells, the spread of Lucifer yellow to neighboring cells was reversibly blocked with 18 α-glycyrrhetinic acid, a gap junction blocker, but it was unaffected by oxidized ATP or probenecid, which block ionotropic ATP-activated channels and organic anion transporters, respectively. Abs against TNF-α significantly reduced the LPS plus IFN-γ-induced increase in dye coupling. In dye-coupled monocytes/macrophages, but not in control cells, both connexin43 protein and mRNA were detected, and their levels were higher in cells with an elevated incidence of dye coupling. In dye-coupled cells, the localization of connexin43 immunoreactivity was diffuse at perinuclear regions and thin cell processes. The addition of 18-α-glycyrrhetinic acid induced a profound reduction of monocyte/macrophage transmigration across a blood brain barrier model. It also induced a significant reduction in the secretion of metalloproteinase-2 in cells treated with TNF-α plus IFN-γ. We propose that some monocyte/macrophage responses are coordinated by connexin-formed membrane channels expressed transiently at inflammatory sites in which these cells form aggregates.
Rules for Gene Usage Inferred from a Comparison of Large-Scale Gene Expression Profiles of T and B Lymphocyte Development
Authors: Hoffmann Reinhard , Bruno Ludovica , Seidl Thomas , Rolink Antonius , Melchers Fritz
Published: April 2014

Ribonucleic acid expression profiles of seven consecutive stages of mouse thymocyte development were generated on high density oligonucleotide arrays. Previously known expression patterns of several genes were confirmed. Ten percent (1,304 of more than 13,000) of the monitored genes were found with 99% confidence to be differentially expressed across all T cell developmental stages. When compared with 1,204 genes differentially expressed in five consecutive B lineage developmental stages of bone marrow, >40% (546 genes) appeared to be shared by both lineages. However, when four pools of functionally distinct cell stages were compared between B and T cell development, DJ-rearranged precursor cells and resting immature precursor cells before and after surface Ag receptor expression shared less than 10%, mature resting lymphocytes between 15 and 20%, and only cycling precursors responding to precursor lymphocyte receptor deposition shared >50% of these differentially expressed genes. Three general rules emerge from these results: 1) proliferation of cells at comparable stages is in majority executed by the same genes; 2) intracellular signaling and intercellular communication are effected largely by different genes; and 3) most genes are not used strictly at comparable, but rather at several, stages, possibly in different functional contexts.
Block in Development at the Pre-B-II to Immature B Cell Stage in Mice Without Igκ and Igλ Light Chain
Authors: Zou Xiangang , Piper Tony A. , Smith Jennifer A. , Allen Nicholas D. , Xian Jian , Brüggemann Marianne
Published: April 2014

Silencing individual C (constant region) λ genes in a κ−/− background reduces mature B cell levels, and L chain-deficient (λ−/−κ−/−) mice attain a complete block in B cell development at the stage when L chain rearrangement, resulting in surface IgM expression, should be completed. L chain deficiency prevents B cell receptor association, and L chain function cannot be substituted (e.g., by surrogate L chain). Nevertheless, precursor cell levels, controlled by developmental progression and checkpoint apoptosis, are maintained, and B cell development in the bone marrow is fully retained up to the immature stage. L chain deficiency allows H chain retention in the cytoplasm, but prevents H chain release from the cell, and as a result secondary lymphoid organs are B cell depleted while T cell levels remain normal.
Suppressor of Cytokine Signaling 1 Inhibits IL-10-Mediated Immune Responses
Authors: Ding Yaozhong , Chen Dongmei , Tarcsafalvi Adel , Su Ruthie , Qin Lihui , Bromberg Jonathan S.
Published: April 2014

IL-10 has proved to be a key cytokine in regulating inflammatory responses by controlling the production and function of various other cytokines. The suppressor of cytokine signaling (SOCS) gene products are a family of cytoplasmic molecules that are essential mediators for negatively regulating cytokine signaling. It has been previously shown that IL-10 induced SOCS3 expression and that forced constitutive expression of SOCS3 inhibits IL-10/STAT3 activation and LPS-induced macrophage activation. In this report, we show that, in addition to SOCS3 expression, IL-10 induces SOCS1 up-regulation in all cell lines tested, including Ba/F3 pro-B cells, MC/9 mast cells, M1 leukemia cells, U3A human fibroblasts, and primary mouse CD4+ T cells. Induction of SOCS molecules is dependent on STAT3 activation by IL-10R1. Cell lines constitutively overexpressing SOCS proteins demonstrated that SOCS1 and SOCS3, but not SOCS2, are able to partially inhibit IL-10-mediated STAT3 activation and proliferative responses. Pretreatment of M1 cells with IFN-γ resulted in SOCS1 induction and a reduction of IL-10-mediated STAT3 activation and cell growth inhibition. IL-10-induced SOCS is associated with the inhibition of IFN-γ signaling in various cell types, and this inhibition is independent of C-terminal serine residues of the IL-10R, previously shown to be required for other anti-inflammatory responses. Thus, the present results show that both SOCS1 and SOCS3 are induced by IL-10 and may be important inhibitors of both IL-10 and IFN-γ signaling. IL-10-induced SOCS1 may directly inhibit IL-10 IFN-γ signaling, while inhibition of other proinflammatory cytokine responses may use additional IL-10R1-mediated mechanisms.
The Shift of Th1 to Th2 Immunodominance Associated with the Chronicity ofMycobacterium bovisBacille Calmette-Guérin Infection Does Not Affect the Memory Response
Authors: Jiao Xinan , Lo-Man Richard , Winter Nathalie , Dériaud Edith , Gicquel Brigitte , Leclerc Claude
Published: April 2014

In the present study we investigated the shaping and evolution of the immunodominance of the T cell response during a chronic mycobacterial infection. Using a recombinant bacille Calmette-Guérin expressing a reporter Ag, the Escherichia coli MalE protein, we analyzed the peptide specificity and the cytokine profile of the T cell response to the reporter Ag by ELISPOT. During the early steps of infection, the T cell response was focused on two dominant MalE epitopes and was characterized by a pure IFN-γ response. Then, in the course of infection the initial IFN-γ response to these two epitopes shifted to a mixed IFN-γ/IL-4 response. At the same time, the peptide specificity of the T cell response was broadened to two additional MalE epitopes characterized by a unique IL-4 response resulting in the establishment of a dominant IL-4 response to the MalE protein at 16 wk postinfection. However, this phenomenon did not impair the outcome of a predominant IFN-γ response upon subsequent MalE recall in vivo performed in the presence of CFA, a Th1-driving adjuvant. These results indicate that the Th2 nature of the immune response established during a chronic infection, which most likely reflects regulatory mechanisms to allow the return to T cell homeostasis, does not shape the Th1/Th2 nature of the memory response.
Lipopolysaccharide (LPS)-Binding Protein Mediates LPS Detoxification by Chylomicrons
Authors: Vreugdenhil Anita C. E. , Rousseau Corine H. , Hartung Thomas , Greve Jan Willem M. , van ‘t Veer Cornelis , Buurman Wim A.
Published: April 2014

Chylomicrons have been shown to protect against endotoxin-induced lethality. LPS-binding protein (LBP) is involved in the inactivation of bacterial toxin by lipoproteins. The current study examined the interaction among LBP, chylomicrons, and bacterial toxin. LBP was demonstrated to associate with chylomicrons and enhance the amount of LPS binding to chylomicrons in a dose-dependent fashion. In addition, LBP accelerated LPS binding to chylomicrons. This LBP-induced interaction of LPS with chylomicrons prevented endotoxin toxicity, as demonstrated by reduced cytokine secretion by PBMC. When postprandial circulating concentrations of chylomicrons were compared with circulating levels of low density lipoprotein, very low density lipoprotein, and high density lipoprotein, chylomicrons exceeded the other lipoproteins in LPS-inactivating capacity. Furthermore, highly purified lipoteichoic acid, an immunostimulatory component of Gram-positive bacteria, was detoxified by incubation with LBP and chylomicrons. In conclusion, our results indicate that LBP associates with chylomicrons and enables chylomicrons to rapidly bind bacterial toxin, thereby preventing cell activation. Besides a role in the detoxification of bacterial toxin present in the circulation, we believe that LBP-chylomicron complexes may be part of a local defense mechanism of the intestine against translocated bacterial toxin.
A Simian Replication-Defective Adenoviral Recombinant Vaccine to HIV-1 Gag
Authors: Fitzgerald Julie C. , Gao Guang-Ping , Reyes-Sandoval Arturo , Pavlakis George N. , Xiang Zhi Q. , Wlazlo Anthony P. , Giles-Davis Wynetta , Wilson James M. , Ertl Hildegund C. J.
Published: April 2014

In animal models, E1-deleted human adenoviral recombinants of the serotype 5 (AdHu5) have shown high efficacy as vaccine carriers for different Ags including those of HIV-1. Humans are infected by common serotypes of human adenovirus such as AdHu5 early in life and a significant percentage has high levels of neutralizing Abs to these serotypes, which will very likely impair the efficacy of recombinant vaccines based on the homologous virus. To circumvent this problem, a novel replication-defective adenoviral vaccine carrier based on an E1-deleted recombinant of the chimpanzee adenovirus 68 (AdC68) was developed. An AdC68 construct expressing a codon-optimized, truncated form of gag of HIV-1 induces CD8+ T cells to gag in mice which at the height of the immune response encompass nearly 20% of the entire splenic CD8+ T cell population. The vaccine-induced immune response provides protection to challenge with a vaccinia gag recombinant virus. Induction of transgene-specific CD8+ T cells and protection against viral challenge elicited by the AdC68 vaccines is not strongly inhibited in animals preimmune to AdHu5 virus. However, the response elicited by the AdHu5 vaccine is greatly attenuated in AdHu5 preimmune animals.
Impaired Clearance of Herpes Simplex Virus Type 1 From Mice Lacking CD1d or NKT Cells Expressing the Semivariant Vα14-Jα281 TCR
Authors: Grubor-Bauk Branka , Simmons Anthony , Mayrhofer Graham , Speck Peter G.
Published: April 2014

Ag-presenting molecule CD1 and CD1-restricted NKT cells are known to contribute to defense against a range of infectious pathogens, including some viruses. CD1-restricted NKT cells, a distinct subpopulation of T cells, have striking and rapid effector functions that contribute to host defense, including rapid production of IFN-γ and IL-4, and activation of NK cells. Consideration of the important contributions of innate and adaptive immunity to clearance of HSV prompted us to investigate the role of CD1 and of NKT cells expressing the Vα14-Jα281 TCR in the pathogenesis of HSV infection. To address this issue, we compared infection in wild-type mice with that in CD1 gene knockout (GKO) and Jα281 GKO mice. In this study, we report impaired clearance of virus and viral Ags, and more florid acute infection in mice lacking CD1 (and by inference, CD1-restricted T cells), in comparison with parental C57BL6 mice. In Jα281 GKO mice there was also impairment of virus clearance, resembling that seen in CD1 GKO mice. These results imply roles for the Vα14-Jα281 subset of NKT cells and for CD1d in control of HSV infection.
CCL28 Has Dual Roles in Mucosal Immunity as a Chemokine with Broad-Spectrum Antimicrobial Activity
Authors: Hieshima Kunio , Ohtani Haruo , Shibano Michiko , Izawa Dai , Nakayama Takashi , Kawasaki Yuri , Shiba Fumio , Shiota Mitsuru , Katou Fuminori , Saito Takuya , Yoshie Osamu
Published: April 2014

CCL28 is a CC chemokine signaling via CCR10 and CCR3 that is selectively expressed in certain mucosal tissues such as exocrine glands, trachea, and colon. Notably, these tissues commonly secrete low-salt fluids. RT-PCR analysis demonstrated that salivary glands expressed CCL28 mRNA at the highest levels among various mouse tissues. Single cells prepared from mouse parotid glands indeed contained a major fraction of CD3−B220low cells that expressed CCR10 at high levels and CCR3 at low levels and responded to CCL28 in chemotaxis assays. Morphologically, these cells are typical plasma cells. By immunohistochemistry, acinar epithelial cells in human and mouse salivary glands were strongly positive for CCL28. Furthermore, human saliva and milk were found to contain CCL28 at high concentrations. Moreover, the C terminus of human CCL28 has a significant sequence similarity to histatin-5, a histidine-rich candidacidal peptide in human saliva. Subsequently, we demonstrated that human and mouse CCL28 had a potent antimicrobial activity against Candida albicans, Gram-negative bacteria, and Gram-positive bacteria. The C-terminal 28-aa peptide of human CCL28 also displayed a selective candidacidal activity. In contrast, CCL27, which is most similar to CCL28 and shares CCR10, showed no such potent antimicrobial activity. Like most other antimicrobial peptides, CCL28 exerted its antimicrobial activity in low-salt conditions and rapidly induced membrane permeability in target microbes. Collectively, CCL28 may play dual roles in mucosal immunity as a chemoattractant for cells expressing CCR10 and/or CCR3 such as plasma cells and also as a broad-spectrum antimicrobial protein secreted into low-salt body fluids.