bluuebunny/crossref_metadata_2025 huggingface dataset:
Key Residues of a Major Cytochrome P4502D6 Epitope Are Located on the Surface of the Molecule
Authors: Ma Yun , Thomas Mark G. , Okamoto Manabu , Bogdanos Dimitrios P. , Nagl Sylvia , Kerkar Nanda , Lopes Agnel R. , Muratori Luigi , Lenzi Marco , Bianchi Francesco B. , Mieli-Vergani Giorgina , Vergani Diego
Published: April 2014

Eukaryotically expressed CYP2D6 is the universal target of liver kidney microsomal Ab type 1 (LKM1) in both type 2 autoimmune hepatitis (AIH) and chronic hepatitis C virus (HCV) infection. In contrast, reactivity to prokaryotically expressed CYP2D6 protein and synthetic peptides is significantly lower in HCV infection than in AIH. The aim of the present study was to characterize LKM1 reactivity against a panel of eukaryotically expressed CYP2D6 constructs in the two conditions. LKM1-positive sera obtained from 16 patients with AIH and 16 with HCV infection were used as probes to perform a complete epitope mapping of CYP2D6. Reactivity to the full-length protein and 16 constructs thereof was determined by radioligand assay. We found that antigenicity is confined to the portion of the molecule C-terminal of aa 193, no reactivity being detectable against the aa sequence 1–193. Reactivity increases stepwise toward the C-terminal in both AIH and HCV, but the frequency of reactivity in the two conditions differs significantly between aa 267–337. To further characterize this region, we introduced a five and a three amino acid swap mutation selected from the homologous regions of CYP2C9 and HCV. This maneuver resulted in a substantial loss of LKM1 binding in both conditions, suggesting that this region contains a major epitope. Molecular modeling revealed that CYP2D6316–327 is exposed on the surface of the protein, and may represent a key target for the autoantibody. These findings provide an initial characterization of the antigenic constitution of the target of LKM1 in AIH and HCV infection.
Negative Feedback Regulation of the Tumor Suppressor PTEN by Phosphoinositide-Induced Serine Phosphorylation
Authors: Birle Diana , Bottini Nunzio , Williams Scott , Huynh Huong , deBelle Ian , Adamson Eileen , Mustelin Tomas
Published: April 2014

The PTEN tumor suppressor phosphatase directly counteracts the multiple functions of phosphatidylinositol 3-kinase by removing phosphate from the D3 position of inositol phospholipids. Like many lymphomas and leukemias, the Jurkat T cell line lacks PTEN protein due to frame-shift mutations in both PTEN alleles and therefore survives in long-term cell culture. We report that PTEN reintroduced into Jurkat was highly phosphorylated on serines 380 and 385 in its C terminus, particularly the former site. Phosphate was also detected at Ser380 in PTEN in untransformed human T cells. Treatments that reduced the levels of D3-phospholipids in the cells resulted in reduced phosphorylation and accelerated degradation of PTEN. In contrast, expression of inactive PTEN-C124G or coexpression of a constitutively active protein kinase B led to increased phosphorylation and slower degradation of PTEN. These results suggest that PTEN normally is subjected to a feedback mechanism of regulation aimed at maintaining homeostatic levels of D3-phosphoinositides, which are crucial for T cell survival and activation.
TCR Engagement Induces Proline-Rich Tyrosine Kinase-2 (Pyk2) Translocation to the T Cell-APC Interface Independently of Pyk2 Activity and in an Immunoreceptor Tyrosine-Based Activation Motif-Mediated Fashion
Authors: Sancho David , Montoya María C. , Monjas Alicia , Gordón-Alonso Mónica , Katagiri Takuya , Gil Diana , Tejedor Reyes , Alarcón Balbino , Sánchez-Madrid Francisco
Published: April 2014

The relocation of kinases in T lymphocytes during their cognate interaction with APCs is essential for lymphocyte activation. We found that the proline-rich tyrosine kinase-2 (Pyk2) is rapidly translocated to the T cell-APC contact area upon T cell-specific recognition of superantigen-pulsed APCs. Stimulation with anti-CD3-coated latex microspheres was sufficient for Pyk2 reorientation, and the coengagement of CD28 boosted Pyk2 redistribution. Nevertheless, Pyk2 translocation did not result in its recruitment to lipid rafts. Two results support that Pyk2 translocation was independent of its kinase activity. First, Lck activity was required for TCR-induced Pyk2 translocation, but not for TCR-induced Pyk2 activation. Second, a kinase-dead Pyk2 mutant was equally translocated upon TCR triggering. In addition, Lck activity alone was insufficient to induce Pyk2 reorientation and activation, requiring the presence of at least one intact immunoreceptor tyrosine-based activation motif (ITAM). Despite the dependence on functional Lck and on phosphorylated ITAM for Pyk2 translocation, the ITAM-binding tyrosine kinase ζ-associated protein 70 (ZAP-70) was not essential. All these data suggest that, by translocating to the vicinity of the immune synapse, Pyk2 could play an essential role in T cell activation and polarized secretion of cytokines.
Essential Role of Extrathymic T Cells in Protection Against Malaria
Authors: Mannoor M. Kaiissar , Halder Ramesh C. , Morshed Sufi Reza M. , Ariyasinghe Anoja , Bakir Hanaa Y. , Kawamura Hiroki , Watanabe Hisami , Sekikawa Hiroho , Abo Toru
Published: April 2014

Athymic nude mice carry neither conventional T cells nor NKT cells of thymic origin. However, NK1.1−TCRint cells are present in the liver and other immune organs of athymic mice, because these lymphocyte subsets are truly of extrathymic origin. In this study, we examined whether extrathymic T cells had the capability to protect mice from malarial infection. Although B6-nu/nu mice were more sensitive to malaria than control B6 mice, these athymic mice were able to survive malaria when a reduced number of parasitized erythrocytes (5 × 103 per mouse) were injected. At the fulminant stage, lymphocytosis occurred in the liver and the major expanding lymphocytes were NK1.1−TCRint cells (IL-2Rβ+TCRαβ+). Unconventional CD8+ NKT cells (Vα14−) also appeared. Similar to the case of B6 mice, autoantibodies (IgM type) against denatured DNA appeared during malarial infection. Immune lymphocytes isolated from the liver of athymic mice which had recovered from malaria were capable of protecting irradiated euthymic and athymic mice from malaria when cell transfer experiments were conducted. In conjunction with the previous results in euthymic mice, the present results in athymic mice suggest that the major lymphocyte subsets associated with protection against malaria might be extrathymic T cells.
Altered Neutrophil Trafficking During Sepsis
Authors: Guo Ren-Feng , Riedemann Niels C. , Laudes Ines J. , Sarma Vidya J. , Kunkel Robin G. , Dilley Kari A. , Paulauskis Joseph D. , Ward Peter A.
Published: April 2014

In sepsis, dysregulation of the inflammatory system is well known, as reflected in excessive inflammatory mediator production, complement activation, and appearance of defects in phagocytic cells. In the current study sepsis was induced in rats by cecal ligation/puncture. Early in sepsis the β1 and β2 integrin content on blood neutrophils increased in a nontranscriptional manner, and the increase in β2, but not β1, integrin content was C5a dependent. Similar changes could be induced in vitro on blood neutrophils following contact with phorbol ester or C5a. Direct injury of lungs of normal rats induced by deposition of IgG immune complexes (IgG-IC) caused 5-fold increases in the myeloperoxidase content that was β2, but not β1, dependent. In contrast, in cecal ligation/puncture lungs myeloperoxidase increased 10-fold after IgG immune complex deposition and was both β1 and β2 integrin dependent. These data suggest that sepsis causes enhanced neutrophil trafficking into the lung via mechanisms that are not engaged in the nonseptic state.
Induction of CD1-Restricted Immune Responses in Guinea Pigs by Immunization with Mycobacterial Lipid Antigens
Authors: Hiromatsu Kenji , Dascher Christopher C. , LeClair Kenneth P. , Sugita Masahiko , Furlong Stephen T. , Brenner Michael B. , Porcelli Steven A.
Published: April 2014

Group 1 CD1 molecules have been shown to present lipid and glycolipid Ags of mycobacteria to human T cells. However, a suitable animal model for the investigation of this component of antimycobacterial immunity has not yet been established. Previously, we found that guinea pigs express multiple isoforms of group 1 CD1 proteins that are homologous to human CD1b and CD1c. In this study, we show that CD1-restricted T cell responses can be generated in guinea pigs following immunization with lipid Ags from Mycobacterium tuberculosis. Splenic T cells from lipid Ag-immunized guinea pigs showed strong proliferative responses to total lipid Ags and partially purified glycolipid fractions from M. tuberculosis. These lipid Ag-reactive T cells were enriched in CD4-negative T cell fractions and showed cytotoxic activity against CD1-expressing guinea pig bone marrow-derived dendritic cells pulsed with M. tuberculosis lipid Ags. Using guinea pig cell lines transfected with individual CD1 isoforms as target cells in cytotoxic T cell assays, we found that guinea pig CD1b and CD1c molecules presented M. tuberculosis glycolipid Ags to T cells raised by mycobacterial lipid immunization. These results were confirmed using a T cell line derived from M. tuberculosis lipid Ag-immunized guinea pigs, which also showed CD1-restricted responses and cytolytic activity. Our results demonstrate that CD1-restricted responses against microbial glycolipid Ags can be generated in vivo by specific immunization and provide support for the use of the guinea pig as a relevant small animal model for the study of CD1-restricted immune responses to mycobacterial pathogens.
Established Human Papillomavirus Type 16-Expressing Tumors Are Effectively Eradicated Following Vaccination with Long Peptides
Authors: Zwaveling Sander , Mota Sandra C. Ferreira , Nouta Jan , Johnson Mark , Lipford Grayson B. , Offringa Rienk , van der Burg Sjoerd H. , Melief Cornelis J. M.
Published: April 2014

Peptide-based vaccines aimed at the induction of effective T cell responses against established cancers have so far only met with limited clinical success and clearly need to be improved. In a preclinical model of human papillomavirus (HPV)16-induced cervical cancer we show that prime-boost vaccinations with the HPV16-derived 35 amino-acid long peptide E743–77, containing both a CTL epitope and a Th epitope, resulted in the induction of far more robust E7-specific CD8+ T cell responses than vaccinations with the minimal CTL epitope only. We demonstrate that two distinct mechanisms are responsible for this effect. First, vaccinations with the long peptide lead to the generation of E7-specific CD4+ Th cells. The level of the induced E7-specific CD8+ T cell response proved to be dependent on the interactions of these Th cells with professional APC. Second, we demonstrate that vaccination with the long peptide and dendritic cell-activating agents resulted in a superior induction of E7-specific CD8+ T cells, even when T cell help was excluded. This suggests that, due to its size, the long peptide was preferably endocytosed, processed, and presented by professional APCs. Moreover, the efficacy of this superior HPV-specific T cell induction was demonstrated in therapeutic prime-boost vaccinations in which the long peptide admixed with the dendritic cell-activating adjuvant oligodeoxynucleotide-CpG resulted in the eradication of large, established HPV16-expressing tumors. Because the vaccine types used in this study are easy to prepare under good manufacturing practice conditions and are safe to administer to humans, these data provide important information for future clinical trials.
The Murine Cytomegalovirus Immunomodulatory Gene m152 Prevents Recognition of Infected Cells by M45-Specific CTL But Does Not Alter the Immunodominance of the M45-Specific CD8 T Cell Response In Vivo
Authors: Gold Marielle C. , Munks Michael W. , Wagner Markus , Koszinowski Ulrich H. , Hill Ann B. , Fling Steven P.
Published: April 2014

Although in vitro studies have shown that herpesviruses, including murine CMV (MCMV), encode genes that interfere with the MHC class I pathway, their effects on the CTL response in vivo is unclear. We identified a Db-restricted CTL epitope from MCMV M45 by screening an MCMV genomic library using CTL clones isolated from mice infected with MCMV lacking m152. Because m152 severely inhibits CTL recognition of M45 in vitro, we questioned whether an M45-specific response would be generated in mice infected with wild-type MCMV expressing m152. Mice infected with wild-type MCMV or MCMVΔm152 made similar responses to the M45 Ag. Moreover, we saw no skewing of the proportion of M45-specific CD8 T cells within the total MCMV-specific response after infection with MCMV with m152. Despite the profound effect m152 has on presentation of M45 in vitro, it does not affect the immunodominance of M45 in the CTL response in vivo.
Modulation of Mac-1 (CD11b/CD18)-Mediated Adhesion by the Leukocyte-Specific Protein 1 Is Key to Its Role in Neutrophil Polarization and Chemotaxis
Authors: Wang Chunjie , Hayashi Hisayoshi , Harrison Rene , Chiu Basil , Chan Jason R. , Ostergaard Hanne L. , Inman Robert D. , Jongstra Jan , Cybulsky Myron I. , Jongstra-Bilen Jenny
Published: April 2014

Leukocyte-specific protein 1 (LSP1) is an intracellular filamentous-actin binding protein which modulates cell motility. The cellular process in which LSP1 functions to regulate motility is not yet identified. In this study, we show that LSP1 negatively regulates fMLP-induced polarization and chemotaxis of neutrophils through its function on adhesion via specific integrins. Using LSP1-deficient (Lsp1−/−) mice, we show increased neutrophil migration into mouse knee joints during zymosan-induced acute inflammation, an inflammatory model in which the number of resident synoviocytes are not affected by LSP1-deficiency. In vitro chemotaxis experiments performed by time-lapse videomicroscopy showed that purified Lsp1−/− bone-marrow neutrophils exhibit an increased migration rate toward a gradient of fMLP as compared with wild-type neutrophils. This difference was observed when cells migrated on fibrinogen, but not fibronectin, suggesting a role for LSP1 in modulating neutrophil adhesion by specific integrins. LSP1 is also a negative regulator of fMLP-induced adhesion to fibrinogen or ICAM-1, but not to ICAM-2, VCAM-1, or fibronectin. These results suggest that LSP1 regulates the function of Mac-1 (CD11b/CD18), which binds only to fibrinogen and ICAM-1 among the substrates we tested. fMLP-induced filamentous actin polarization is also increased in the absence of LSP1 when cells were layered on fibrinogen, but not on fibronectin. Our findings suggest that the increased neutrophil recruitment in Lsp1−/− mice during acute inflammation derives from the negative regulatory role of LSP1 on neutrophil adhesion, polarization, and migration via specific integrins, such as Mac-1, which mediate neutrophil responses to chemotactic stimuli.
Differing Activities of Homeostatic Chemokines CCL19, CCL21, and CXCL12 in Lymphocyte and Dendritic Cell Recruitment and Lymphoid Neogenesis
Authors: Luther Sanjiv A. , Bidgol Afshin , Hargreaves Diana C. , Schmidt Andrea , Xu Ying , Paniyadi Jyothi , Matloubian Mehrdad , Cyster Jason G.
Published: April 2014

Despite their widespread expression, the in vivo recruitment activities of CCL19 (EBV-induced molecule 1 ligand chemokine) and CXCL12 (stromal cell-derived factor 1) have not been established. Furthermore, although CXCL13 (B lymphocyte chemoattractant) has been shown to induce lymphoid neogenesis through induction of lymphotoxin (LT)α1β2, it is unclear whether other homeostatic chemokines have this property. In this work we show that ectopic expression in pancreatic islets of CCL19 leads to small infiltrates composed of lymphocytes and dendritic cells and containing high endothelial venules and stromal cells. Ectopic CXCL12 induced small infiltrates containing few T cells but enriched in dendritic cells, B cells, and plasma cells. Comparison of CCL19 transgenic mice with mice expressing CCL21 (secondary lymphoid tissue chemokine) revealed that CCL21 induced larger and more organized infiltrates. A more significant role for CCL21 is also suggested in lymphoid tissues, as CCL21 protein was found to be present in lymph nodes and spleen at much higher concentrations than CCL19. CCL19 and CCL21 but not CXCL12 induced LTα1β2 expression on naive CD4 T cells, and treatment of CCL21 transgenic mice with LTβR-Fc antagonized development of organized lymphoid structures. LTα1β2 was also induced on naive T cells by the cytokines IL-4 and IL-7. These studies establish that CCL19 and CXCL12 are sufficient to mediate cell recruitment in vivo and they indicate that LTα1β2 may function downstream of CCL21, CCL19, and IL-2 family cytokines in normal and pathological lymphoid tissue development.
IL-15-Dependent Activation-Induced Cell Death-Resistant Th1 Type CD8αβ+NK1.1+ T Cells for the Development of Small Intestinal Inflammation
Authors: Ohta Noriyuki , Hiroi Takachika , Kweon Mi-Na , Kinoshita Naotoshi , Jang Myoung Ho , Mashimo Tadashi , Miyazaki Jun-Ichi , Kiyono Hiroshi
Published: April 2014

To clarify the role of IL-15 at local sites, we engineered a transgenic (Tg) mouse (T3b-IL-15 Tg) to overexpress human IL-15 preferentially in intestinal epithelial cells by the use of T3b-promoter. Although IL-15 was expressed in the entire small intestine (SI) and large intestines of the Tg mice, localized inflammation developed in the proximal SI only. Histopathologic study revealed reduced villus length, marked infiltration of lymphocytes, and vacuolar degeneration of the villus epithelium, beginning at ∼3–4 mo of age. The numbers of CD8+ T cells, especially CD8αβ+ T cells expressing NK1.1, were dramatically increased in the lamina propria of the involved SI. The severity of inflammation corresponded to increased numbers of CD8αβ+NK1.1+ T cells and levels of production of the Th1-type cytokines IFN-γ and TNF-α. Locally overexpressed IL-15 was accompanied by increased resistance of CD8αβ+ NK1.1+ T cells to activation-induced cell death. Our results suggest that chronic inflammation in the SI in this murine model is mediated by dysregulation of epithelial cell-derived IL-15. The model may contribute to understanding the role of CD8+ T cells in human Crohn’s disease involving the SI.
Disruption of a SinglePtenAllele Augments the Chemotactic Response of B Lymphocytes to Stromal Cell-Derived Factor-1
Authors: Fox Joanne A. , Ung Karen , Tanlimco Sonia G. , Jirik Frank R.
Published: April 2014

The tumor suppressor, Pten, has emerged as a critical negative regulator of phosphatidylinositol-3-kinase-dependent intracellular signaling pathways responsible for phenomena such as cellular adhesion, proliferation, and apoptosis. Herein, we present evidence that Pten regulates chemokine-dependent events in B lymphocytes. Primary B cells isolated from Pten+/− mice demonstrated increased responsiveness to stromal cell-derived factor-1-induced chemotaxis. This was accompanied by an elevated level of protein kinase B phosphorylation on Ser473. Our results suggest not only that Pten may be an important regulator of stromal cell-derived factor-1-directed chemotaxis, but also that Pten heterozygosity is associated with increased cellular sensitivity to this chemokine, likely via dysregulation of events lying downstream of phosphatidylinositol-3-kinase. These observations suggest a mechanism by which loss of a single Pten allele may confer a selective advantage on cells during multistep tumor progression.
Inhibition of Endogenous TGF-β During Experimental Osteoarthritis Prevents Osteophyte Formation and Impairs Cartilage Repair
Authors: Scharstuhl Alwin , Glansbeek Harrie L. , van Beuningen Henk M. , Vitters Elly L. , van der Kraan Peter M. , van den Berg Wim B.
Published: April 2014

Osteoarthritis has as main characteristics the degradation of articular cartilage and the formation of new bone at the joint edges, so-called osteophytes. In this study enhanced expression of TGF-β1 and -β3 was detected in developing osteophytes and articular cartilage during murine experimental osteoarthritis. To determine the role of endogenous TGF-β on osteophyte formation and articular cartilage, TGF-β activity was blocked via a scavenging soluble TGF-β-RII. Our results clearly show that inhibition of endogenous TGF-β nearly completely prevented osteophyte formation. In contrast, treatment with recombinant soluble TGF-β-RII markedly enhanced articular cartilage proteoglycan loss and reduced the thickness of articular cartilage. In conclusion, we show for the first time that endogenous TGF-β is a crucial factor in the process of osteophyte formation and has an important function in protection against cartilage loss.
An Autoreactive γδ TCR Derived from a Polymyositis Lesion
Authors: Wiendl Heinz , Malotka Joachim , Holzwarth Brigitte , Weltzien Hans-Ulrich , Wekerle Hartmut , Hohlfeld Reinhard , Dornmair Klaus
Published: April 2014

To investigate the role of γδ T cells in human autoimmune disease we expressed and characterized a γδ TCR from an autoimmune tissue lesion. The TCR was first identified in a rare form of polymyositis characterized by a monoclonal infiltrate of γδ T cells which invaded and destroyed skeletal muscle fibers. The Vγ1.3-Jγ1-Cγ1/Vδ2-Jδ3 TCR cDNA of the original muscle invasive γδ T cell clone was reconstructed from unrelated cDNA and transfected into the mouse hybridoma BW58α−β−. Appropriate anti-human γδ TCR Abs stimulated the TCR transfectants to produce IL-2, thus demonstrating that the human γδ TCR functionally interacted with murine signaling components. The transfected Vγ1.3/Vδ2 TCR recognized a cytosolic protein expressed in cultured human myoblasts and TE671 rhabdomyosarcoma cells. The Ag was recognized in the absence of presenting cells. Using a panel of control γδ TCR transfectants with defined exchanges in different positions of both TCR chains, we showed that the γδ TCR recognized its Ag in a TCR complementarity-determining region 3-dependent way. To our knowledge, this is the first example of a molecularly defined γδ TCR directly derived from an autoimmune tissue lesion. The strategy used in this study may be applicable to other autoimmune diseases.
Antimelanoma Activity of CTL Generated from Peripheral Blood Mononuclear Cells After Stimulation with Autologous Dendritic Cells Pulsed with Melanoma gp100 Peptide G209-2M Is Correlated to TCR Avidity
Authors: Yang Sixun , Linette Gerald P. , Longerich Simonne , Haluska Frank G.
Published: April 2014

Anchor residue-modified peptides derived from tumor-associated Ag have demonstrated success in engendering immune responses in clinical studies. However, tumor regression does not always correlate with immune responses. One hypothesis to explain this is that CTL resulting from such immunization approaches are variable in antitumor potency. In the present study, we evaluated this hypothesis by characterizing the activity of tumor-associated Ag-specific CTL. We chose an anchor residue-modified peptide from gp100, G209-2M, and used peptide-pulsed dendritic cells to generate CTL from PBMC of HLA-A2+ normal donors. The specificities and avidities of the resulting CTL were evaluated. The results demonstrate that CTL generated by G209-2M can be classified into three categories: G209-2M-specific CTL which are cytotoxic only to G209-2M-pulsed targets; peptide-specific CTL which recognize both G209 and G209-2M peptides but not melanomas; and melanoma-reactive CTL which recognize peptide-pulsed targets as well as HLA-A2+gp100+ melanomas. CTL that kill only peptide-pulsed targets require a higher peptide concentration to mediate target lysis, whereas CTL that lyse melanomas need a lower peptide concentration. Increasing peptide density on melanomas by loading exogenous G209 peptide enhances their sensitivity to peptide-specific CTL. High avidity CTL clones also demonstrate potent antimelanoma activity in melanoma model in nude mice. Injection of G209 peptide around transplanted tumors significantly enhances the antitumor activity of low avidity CTL. These results suggest that peptide stimulation causes expansion of T cell populations with a range of avidities. Successful immunotherapy may require selective expansion of the higher-avidity CTL and intratumor injection of the peptide may enhance the effect of peptide vaccines.
Differential Induction of Mucosal and Systemic Antibody Responses in Women After Nasal, Rectal, or Vaginal Immunization: Influence of the Menstrual Cycle
Authors: Kozlowski Pamela A. , Williams Selvi B. , Lynch Rebecca M. , Flanigan Timothy P. , Patterson Rosalyn R. , Cu-Uvin Susan , Neutra Marian R.
Published: April 2014

A cholera vaccine containing killed vibrios and cholera toxin B subunit (CTB) was used to compare mucosal immunization routes for induction of systemic and mucosal Ab. Four groups of women were given three monthly immunizations by the rectal immunization (Rimm) route, nasal immunization (Nimm) route, or vaginal immunization route during either the follicular (V-FPimm) or luteal (V-LPimm) menstrual cycle phase. Nimm was performed with 10-fold less vaccine to determine if administration of less Ag by this route can, as in rodents, produce mucosal Ab responses comparable to those induced by higher dose Rimm or vaginal immunization. Concentrations of Ab induced in sera and secretions were measured by ELISA. None of these routes produced durable salivary Ab responses. Nimm induced greatest levels of CTB-specific IgG in sera. Rimm failed to generate CTB-specific IgA in genital tract secretions. Nimm, V-FPimm, and V-LPimm all produced cervical CTB-specific IgA responses comparable in magnitude and frequency. However, only V-FPimm induced cervical IgA2-restricted Ab to the bacterial LPS vaccine component. V-FPimm, but not V-LPimm, also induced CTB-specific IgA in rectal secretions. Nimm was superior to V-FPimm for producing rectal CTB-specific IgA, but the greatest amounts of CTB-specific IgA and LPS-specific IgA, IgG, and IgM Ab were found in rectal secretions of Rimm women. These data suggest that in women, Nimm alone could induce specific Ab in serum, the genital tract, and rectum. However, induction of genital tract and rectal Ab responses of the magnitude generated by local V-FPimm or Rimm will likely require administration of comparably high nasal vaccine dosages.
Induction of Autoantibody Production Is Limited in Nonautoimmune Mice
Authors: Singh Ram Raj , Ebling Fanny M. , Albuquerque D. Alves , Saxena Vijay , Kumar Vipin , Giannini Edward H. , Marion Tony N. , Finkelman Fred D. , Hahn Bevra H.
Published: April 2014

Many individuals develop a single or a few brief episodes of autoimmunity from which they recover. Mechanisms that quell pathologic autoimmunity following such a breakdown of self-tolerance are not clearly understood. In this study, we show that in nonautoimmune mice, dsDNA-specific autoreactive B cells exist but remain inactive. This state of inactivation in dsDNA-specific B cells could be disrupted by autoreactive Th cells; in this case T cells that react with peptides from the VH region of anti-DNA Abs (hereafter called anti-VH T cells). Immunization with anti-DNA mAb, its γ-chain or peptides derived from its VH region induced anti-VH Th cells, IgG anti-dsDNA Ab, and proteinuria. The breakdown of B cell tolerance in nonautoimmune mice, however, was short-lived: anti-DNA Ab and nephritis subsided despite subsequent immunizations. The recovery from autoimmunity temporally correlated with the appearance of T cells that inhibited anti-DNA Ab production. Such inhibitory T cells secreted TGFβ; the inhibition of anti-DNA Ab production by these cells was partly abolished by anti-TGFβ Ab. Even without immunization, nonautoimmune mice possess T cells that can inhibit autoantibody production. Thus, inhibitory T cells in nonautoimmune mice may normally inhibit T-dependent activation of autoreactive B cells and/or reverse such activation following stimulation by Th cells. The induction of such inhibitory T cells may play a role in protecting nonautoimmune mice from developing chronic autoimmunity.
CD123bright Plasmacytoid Predendritic Cells: Progenitors Undergoing Cell Fate Conversion?
Authors: Comeau Michael R. , Van der Vuurst de Vries Anne-Renee , Maliszewski Charles R. , Galibert Laurent
Published: April 2014

CD123bright plasmacytoid cells (PC) and CD1c+ peripheral blood myeloid dendritic cells (DC) are two human DC precursors that can be expanded in vivo by Fms-like tyrosine kinase 3 ligand (FL). It has been proposed that PC and myeloid CD1c+ DC may represent two distinct lineages of DC. However, the phylogenetic affiliation of PC and its relationship with myeloid DC remain controversial. Here we show that CD123brightHLA-DR+ PC from FL-treated healthy volunteers can be divided into mutually exclusive subsets that harbor either lymphoid or myeloid features. Lymphoid-like PC represent the majority of PC and include pTα-, CD3ε-, and CD7-expressing cells. They exhibit TCR-β gene loci in germline configuration and show low allostimulatory capacity, but produce type I IFN upon virus infection and can be differentiated in vitro into potent APC. Myeloid-like PC represent a minor fraction of the total PC population. They exhibit a striking PC/myeloid DC intermediate phenotype (CD5+CD11clowCD45RAlowCD45RO−CD101+), produce proinflammatory cytokines, and do not require in vitro maturation to act as potent APCs. We propose that, rather than forming a lineage, PC might represent a population of lymphoid cells undergoing an in vivo cell fate conversion from a lymphoid to a myeloid cell type.
Cutting Edge: Fever-Associated Temperatures Enhance Neutrophil Responses to Lipopolysaccharide: A Potential Mechanism Involving Cell Metabolism
Authors: Rosenspire Allen J. , Kindzelskii Andrei L. , Petty Howard R.
Published: April 2014

Although much progress has been made in elucidating the mechanisms underlying the physiological regulation of fever, there is little understanding of the biological utility of fever’s thermal component. Considering the evolutionary co-conservation of fever and innate immunity, we hypothesize that fever’s thermal component might in general augment innate immune function and, in particular, neutrophil activation. Accordingly, we have evaluated the effect of febrile temperatures on neutrophil function at the single-cell level. We find that reactive oxygen intermediates and NO release are greatly enhanced at febrile temperatures for unstimulated as well as LPS-stimulated adherent human neutrophils. Furthermore, our studies suggest that these changes in oxidant release are linked to upstream changes in NADPH dynamics. Inasmuch as reactive oxygen intermediates and NO production are important elements in innate immune responses to bacterial pathogens, we suggest that the febrile rise in core temperature is a broad-based systemic signaling mechanism to enhance innate immunity.
Sonic Hedgehog Signaling Modulates Activation of and Cytokine Production by Human Peripheral CD4+ T Cells
Authors: Stewart Gareth A. , Lowrey Jacqueline A. , Wakelin Sonia J. , Fitch Paul M. , Lindey Susannah , Dallman Margaret J. , Lamb Jonathan R. , Howie Sarah E. M.
Published: April 2014

Sonic hedgehog (Shh) is important in the growth and differentiation of a variety of cell types, including the development of T cells in the thymus. This prompted us to investigate whether Shh signaling is a functional component of the physiological response of human mature CD4+ T cells following Ag recognition. In this study, we demonstrate that Shh and its receptor Patched (Ptc) are expressed on resting and activated human peripheral CD4+ T cells. In approximately one-half of the randomly selected, anonymous blood donors tested, exposure of anti-CD3/28 Ab-activated CD4+ T cells to the biologically active N-terminal Shh peptide increased the transcription of ptc, thereby demonstrating that Shh signaling had occurred. Furthermore, the addition of exogenous Shh amplified the production of IL-2, IFN-γ, and IL-10 by activated CD4+ T cells. The synthesis of IL-2 and IFN-γ, but not IL-10, by CD4+ T cells was down-regulated by the addition of neutralizing anti-Shh Ab. Cell surface expression of CD25 and CD69 on activated T cells was up-regulated by exogenous Shh, whereas in the presence of the neutralizing anti-Shh Ab expression it was reduced. Collectively, our findings demonstrate that Shh-mediated signaling is a physiological component of T cell responses, which acts to modulate CD4+ T cell effector function.
Antigen-Nonspecific Recruitment of Th2 Cells to the Lung as a Mechanism for Viral Infection-Induced Allergic Asthma
Authors: Stephens Robin , Randolph David A. , Huang Guangming , Holtzman Michael J. , Chaplin David D.
Published: April 2014

Respiratory viral infections have been shown to trigger exacerbations of asthma; however, the mechanism by which viral Th1-type inflammation exacerbates an allergic Th2-type disease remains unclear. We have previously shown that although adoptively transferred Th2 cells are inefficiently recruited to the lung in response to Ag, cotransfer of Th1 cells can increase accumulation of Th2 cells. In this study, we show that respiratory viral infection increases recruitment of resting Th2 cells specific for OVA even in the absence of OVA challenge. These findings suggest that the mechanism by which Th1-type inflammation enhances allergy is via an effect on recruitment. To study the role of the antigenic specificity of Th1 cells in the enhancement of Th2 cell recruitment and to determine whether virus-induced recruitment of OVA-specific Th2 cells may involve Th1 cells specific to a different Ag, we tested whether hen egg lysozyme-specific Th1 cells could synergize with OVA-specific Th2 cells. Challenge of mice that had received adoptively transferred Th1 cells plus Th2 cells induced the expression of inflammatory chemokines in the lung and increased both recruitment and activation of Th2 cells, leading to eosinophil recruitment, even in the absence of challenge with the Th2 Ag. Interestingly, as IL-5 supports eosinophilia, culture of resting Th2 cells with fresh APC induced production of IL-5 in the absence of specific Ag. Thus, Ag-specific activation of Th1 cells enhances the recruitment potential of the lung leading to recruitment and activation of Th2 cells. This implies that circulating Th2 cells in allergic individuals could enter the lungs in response to infection or inflammation and become activated to trigger allergy.
The Osmoprotective Function of the NFAT5 Transcription Factor in T Cell Development and Activation
Authors: Trama Jason , Go William Y. , Ho Steffan N.
Published: April 2014

The NFAT5/TonEBP transcription factor, a recently identified rel/NF-κB family member, activates transcription of osmocompensatory genes in response to extracellular hyperosmotic stress. However, the function of NFAT5 under isosmotic conditions present in vivo remains unknown. Here we demonstrate that NFAT5 is necessary for optimal T cell development in vivo and allows for optimal cell growth ex vivo under conditions associated with osmotic stress. Transgenic mice expressing an inhibitory form of NFAT5 in developing and mature T cells exhibited a 30% reduction in thymic cellularity evenly distributed among thymic subsets, consistent with the uniform expression and nuclear localization of NFAT5 in each subset. This was associated with a 25% reduction in peripheral CD4+ T cells and a 50% reduction in CD8+ T cells. While transgenic T cells exhibited no impairment in cell growth or cytokine production under normal culture conditions, impaired cell growth was observed under both hyperosmotic conditions and isosmotic conditions associated with osmotic stress. Transgenic thymocytes also demonstrated increased sensitivity to osmotic stress. Consistent with this, the system A amino acid transporter gene ATA2 exhibited NFAT5 dependence under hypertonic conditions but not in response to amino acid deprivation. Expression of the TNF-α gene, a putative NFAT5 target, was not altered in transgenic T cells. These results not only demonstrate an osmoprotective function for NFAT5 in primary cells but also show that NFAT5 is necessary for optimal thymic development in vivo, suggesting that developing thymocytes within the thymic microenvironment are subject to an osmotic stress that is effectively countered by NFAT5-dependent responses.
Lack of Effector Cell Function and Altered Tetramer Binding of Tumor-Infiltrating Lymphocytes
Authors: Blohm Ulrike , Roth Evelyn , Brommer Kathrin , Dumrese Tilman , Rosenthal Felicia M. , Pircher Hanspeter
Published: April 2014

Tumor-specific CD8 T cell responses to MCA102 fibrosarcoma cells expressing the cytotoxic T cell epitope gp33 from lymphocytic choriomeningitis virus were studied. MCA102gp33 tumors grew progressively in C57BL/6 mice, despite induction of peripheral gp33-tetramer+ T cells that were capable of mediating antiviral protection, specific cell rejection, and concomitant tumor immunity. MCA102gp33 tumors were infiltrated with a high number (∼20%) of CD11b+CD11c− macrophage-phenotype cells that were able to cross-present the gp33 epitope to T cells. Tumor-infiltrating CD8 T cells exhibited a highly activated phenotype but lacked effector cell function. Strikingly, a significant portion of tumor-infiltrating lymphocytes expressed TCRs specific for gp33 but bound MHC tetramers only after cell purification and a 24-h resting period in vitro. The phenomenon of “tetramer-negative T cells” was not restricted to tumor-infiltrating lymphocytes from MCA102gp33 tumors, but was also observed when Ag-specific T cells derived from an environment with high Ag load were analyzed ex vivo. Thus, using a novel tumor model, allowing us to trace tumor-specific T cells at the single cell level in vivo, we demonstrate that the tumor microenvironment is able to alter the functional activity of T cells infiltrating the tumor mass.
Release and Intercellular Transfer of Cell Surface CD81 Via Microparticles
Authors: Fritzsching Benedikt , Schwer Björn , Kartenbeck Jürgen , Pedal Angelika , Horejsi Vaclav , Ott Melanie
Published: April 2014

The human tetraspan molecule CD81 is a coreceptor in B and T cell activation and a candidate receptor for hepatitis C virus infection. We examined the surface expression of CD81 on B and T lymphocytes by quantitative flow cytometry. Upon cellular activation, CD81 surface levels were rapidly reduced. This reduction occurred as early as 1 h after activation and was linked to the release of CD81-positive microparticles into the cell culture medium. CD81 mRNA levels were not affected early after activation, but the release of CD81-positive microparticles was rapidly enhanced. In addition, intercellular transfer of CD81 was observed upon coculture of CD81-positive donor cells (Jurkat T cell line) with CD81-negative acceptor cells (U937 promonocytic cell line). This transfer was rapidly increased upon T cell activation, coinciding with enhanced CD81 release from activated Jurkat cells. We propose that the release and intercellular trafficking of CD81-positive microparticles regulate the expression of CD81 surface receptors in lymphocytes and play a role in the immune response during infections.
Expression of Programmed Death 1 Ligands by Murine T Cells and APC
Authors: Yamazaki Tomohide , Akiba Hisaya , Iwai Hideyuki , Matsuda Hironori , Aoki Mami , Tanno Yuka , Shin Tahiro , Tsuchiya Haruo , Pardoll Drew M. , Okumura Ko , Azuma Miyuki , Yagita Hideo
Published: April 2014

Programmed death 1 (PD-1) is a new member of the CD28/CTLA-4 family, which has been implicated in the maintenance of peripheral tolerance. Two ligands for PD-1, namely, B7-H1 (PD-L1) and B7-DC (PD-L2), have recently been identified as new members of the B7 family but their expression at the protein level remains largely unknown. To characterize the expression of B7-H1 and B7-DC, we newly generated an anti-mouse B7-H1 mAb (MIH6) and an anti-mouse B7-DC mAb (TY25). MIH6 and TY25 immunoprecipitated a single molecule of 43 and 42 kDa from the lysate of B7-H1 and B7-DC transfectants, respectively. Flow cytometric analysis revealed that B7-H1 was broadly expressed on the surface of mouse tumor cell lines while the expression of B7-DC was rather restricted. PD-1 was expressed on anti-CD3-stimulated T cells and anti-IgM plus anti-CD40-stimulated B cells at high levels but was undetectable on activated macrophages or DCs. B7-H1 was constitutively expressed on freshly isolated splenic T cells, B cells, macrophages, and dendritic cells (DCs), and up-regulated on T cells by anti-CD3 stimulation on macrophages by LPS, IFN-γ, GM-CSF, or IL-4, and on DCs by IFN-γ, GM-CSF, or IL-4. In contrast, B7-DC expression was only inducible on macrophages and DCs upon stimulation with IFN-γ, GM-CSF, or IL-4. The inducible expression of PD-1 ligands on both T cells and APCs may suggest new paradigms of PD-1-mediated immune regulation.
G Protein-Coupled Chemokine Receptors Induce Both Survival and Apoptotic Signaling Pathways
Authors: Vlahakis Stacey R. , Villasis-Keever Angelina , Gomez Timothy , Vanegas Maria , Vlahakis Nicholas , Paya Carlos V.
Published: April 2014

Chemokine receptors are essential for triggering chemotaxis to immune cells; however, a number of them can also mediate death when engaged by nonchemokine ligands. When the chemokine receptor CXCR4 is engaged by stromal cell-derived factor (SDF1)α, it triggers cells to chemotax, and in some cell types such as neurons, causes cell death. To elucidate this dual and opposing receptor function, we have investigated whether CXCR4 activation by its chemokine SDF1α could lead to the simultaneous activation of both anti- and proapoptotic signaling pathways; the balance ultimately influencing cell survival. CXCR4 activation in CD4 T cells by SDF1α led to the activation of the prosurvival second messengers, Akt and extracellular signal-regulated protein kinase. Selective inhibition of each signal demonstrated that extracellular signal-regulated protein kinase is essential for mediating SDF1α-triggered chemotaxis but does not confer an antiapoptotic state. In contrast, Akt activation through CXCR4 by SDF1α interactions is necessary to confer resistance to apoptosis. The proapoptotic signaling pathway triggered by SDF1α-CXCR4 interaction involves the Giα protein-independent activation of the proapoptotic MAPK (p38). Furthermore, other chemokines and chemokine receptors also signal chemotaxis and proapoptotic effects via similar pathways. Thus, Giα protein-coupled chemokine receptors can function as death prone receptors and the balance between the above signaling pathways will ultimately mandate the fate of the activated cell.
Retinoic Acid Stimulates the Cell Cycle Machinery in Normal T Cells: Involvement of Retinoic Acid Receptor-Mediated IL-2 Secretion
Authors: Ertesvag Aase , Engedal Nikolai , Naderi Soheil , Blomhoff Heidi Kiil
Published: April 2014

The mechanisms whereby vitamin A stimulates the immune system are poorly understood. In the current study, we attempted to elucidate the potential mechanisms of action of all-trans retinoic acid (atRA) on proliferation of human T lymphocytes. We found that physiological levels of atRA potently augmented T cell proliferation when added in combination with common T cell-stimulating agents. This was reflected in a time- and concentration-dependent stimulation of the cell cycle machinery. The presence of atRA led to elevated levels of cyclin D3, -E, and -A, decreased levels of p27Kip1, increased activity of cyclin-dependent kinase 2, and enhanced phosphorylation of the retinoblastoma protein (pRB). The atRA-mediated changes in the cell cycle machinery were late events, appearing after 20 h of stimulation, indicating that the effects of atRA were indirect. atRA did not alter the expression of the high-affinity IL-2R. However, the level of IL-2 secreted by T cells was strongly enhanced by atRA. rIL-2 was able to substitute for the effects of atRA on the cell cycle machinery and on DNA synthesis, and blocking the IL-2R markedly inhibited atRA-induced cell proliferation and pRB phosphorylation. A retinoic acid receptor (RAR)-selective agonist and 9-cis-RA had the same potency as atRA on T cell proliferation and IL-2 secretion, whereas a retinoid X receptor-selective agonist had only marginal effects. Furthermore, a RAR-selective antagonist completely suppressed T cell proliferation and pRB phosphorylation induced by atRA. Taken together, these results suggest that atRA stimulates the cell cycle machinery and proliferation of normal human T cells by increasing IL-2 secretion through mechanisms involving RARs.
Immune Tolerance to Combined Organ and Bone Marrow Transplants After Fractionated Lymphoid Irradiation Involves Regulatory NK T Cells and Clonal Deletion
Authors: Higuchi Masanori , Zeng Defu , Shizuru Judith , Gworek Jennifer , Dejbakhsh-Jones Sussan , Taniguchi Masaru , Strober Samuel
Published: April 2014

Immune tolerance to organ transplants has been reported in laboratory animals and in humans after nonmyeloablative conditioning of the host and infusion of donor bone marrow cells. We examined the mechanisms of immune tolerance to mouse cardiac allografts in MHC-mismatched hosts that developed mixed chimerism after posttransplant conditioning with a 2-wk course of multiple doses of lymphoid tissue irradiation, depletive anti-T cell Abs, and an infusion of donor bone marrow cells. When CD1−/− or Jα281−/− hosts with markedly reduced NK T cells were used instead of wild-type hosts, then the conditioning regimen failed to induce tolerance to the heart allografts despite the development of mixed chimerism. Tolerance could be restored to the CD1−/− hosts by infusing enriched T cells from the bone marrow of wild-type mice containing CD1-reactive T cells but not from CD1−/− host-type mice. Tolerance could not be induced in either IL-4−/− or IL-10−/− hosts given the regimen despite the development of chimerism and clonal deletion of host T cells to donor MHC-Ags in the IL-10−/− hosts. We conclude that immune tolerance to bone marrow transplants involves clonal deletion, and tolerance to heart allografts in this model also involves regulatory CD1-reactive NK T cells.
Human MHC Class I Transgenic Mice Deficient for H2 Class I Expression Facilitate Identification and Characterization of New HLA Class I-Restricted Viral T Cell Epitopes
Authors: Cheuk Eve , D’Souza Celine , Hu Ningjie , Liu Youan , Lang Haili , Chamberlain John W.
Published: April 2014

Although mice transgenic (Tg) for human MHC (HLA) class I alleles could provide an important model for characterizing HLA-restricted viral and tumor Ag CTL epitopes, the extent to which Tg mouse T cells become HLA restricted in the presence of endogenous H2 class I and recognize the same peptides as in HLA allele-matched humans is not clear. We previously described Tg mice carrying the HLA-B27, HLA-B7, or HLA-A2 alleles expressed as fully native (HLAnat) (with human β2-microglobulin) and as hybrid human/mouse (HLAhyb) molecules on the H2b background. To eliminate the influence of H2b class I, each HLA Tg strain was bred with a H2-Kb/H2-Db-double knockout (DKO) strain to generate mice in which the only classical class I expression was the human molecule. Expression of each HLAhyb molecule and HLA-B27nat/human β2-microglobulin led to peripheral CD8+ T cell levels comparable with that for mice expressing a single H2-Kb or H2-Db gene. Influenza A infection of Tg HLA-B27hyb/DKO generated a strong CD8+ T cell response directed at the same peptide (flu nucleoprotein NP383–391) recognized by CTLs from flu-infected B27+ humans. As HLA-B7/flu epitopes were not known from human studies, we used flu-infected Tg HLA-B7hyb/DKO mice to examine the CTL response to candidate peptides identified based on the B7 binding motif. We have identified flu NP418–426 as a major HLA-B7-restricted flu CTL epitope. In summary, the HLA class I Tg/H2-K/H2-D DKO mouse model described in this study provides a sensitive and specific approach for identifying and characterizing HLA-restricted CTL epitopes for a variety of human disease-associated Ags.