bluuebunny/crossref_metadata_2025 huggingface dataset:
Cross-Linking of CD32 Induces Maturation of Human Monocyte-Derived Dendritic Cells Via NF-κB Signaling Pathway
Authors: Bánki Zoltán , Kacani Laco , Müllauer Brigitte , Wilflingseder Doris , Obermoser Gerlinde , Niederegger Harald , Schennach Harald , Sprinzl Georg M. , Sepp Norbert , Erdei Anna , Dierich Manfred P. , Stoiber Heribert
Published: April 2014

Dendritic cells (DC) represent a unique set of APCs that initiate immune responses through priming of naive T cells. Maturation of DC is a crucial step during Ag presentation and can be induced by triggering a broad spectrum of DC surface receptors. Although human DC express several receptors for the Fc portion of IgG which were described to play an important role in Ag internalization, little is known about the effects of IgG or immune complexes on DC maturation. In this study, we show that cross-linking of FcγR-type II (CD32) with immobilized IgG (imIgG) can induce maturation of human monocyte-derived DC via the NF-κB signaling pathway. IgG-mediated maturation was accompanied by a moderate increase of IL-10 secretion, whereas no IL-12 production was observed. Involvement of CD32 was further supported by experiments with the anti-CD32 mAb, which blocked IgG-triggered DC maturation and cytokine secretion significantly. Furthermore, DC cultivated in the presence of imIgG induced allogeneic T cell proliferation. Because this imIgG-induced maturation was considerably impaired in monocyte-derived DC from systemic lupus erythematosus patients, we suggest that DC, which matured in the presence of immune complexes, may contribute to prevention of pathological immune responses.
LIGHT (a Cellular Ligand for Herpes Virus Entry Mediator and Lymphotoxin Receptor)-Mediated Thymocyte Deletion Is Dependent on the Interaction Between TCR and MHC/Self-Peptide
Authors: Wang Jing , Fu Yang-Xin
Published: April 2014

Negative selection serves as a major mechanism to maintain self-tolerance. We previously reported that LIGHT (a cellular ligand for herpes virus entry mediator and lymphotoxin receptor), a TNF family member, plays an important role in thymocyte development via promoting apoptosis of double-positive thymocytes. Here, we demonstrated that LIGHT-mediated deletion of thymocyte requires the strong interaction of TCR with MHC/self-peptide. Transgenic mice overexpressing LIGHT in thymocytes were bred with a transgenic mouse line expressing a TCR recognizing the H-Y male Ag in the context of H-2b class I MHC molecules. In male H-Y/LIGHT double-transgenic mice, more efficient negative selection of H-Y T cells occurred, and total thymocyte number was further reduced compared with H-Y/negative littermates. In contrast, the presence of LIGHT transgene had no evident impact on the thymocyte development of female H-Y/LIGHT double-transgenic mice. Taken together, LIGHT plays a role in negative selection of thymocytes via inducing the apoptosis of thymocytes bearing high affinity TCR during negative selection.
Detection and Characterization of T Cells Specific for BDC2.5 T Cell-Stimulating Peptides
Authors: You Sylvaine , Chen Cyndi , Lee Wen-Hui , Wu Chun-Hua , Judkowski Valeria , Pinilla Clemencia , Wilson Darcy B. , Liu Chih-Pin
Published: April 2014

Nonobese diabetic (NOD) mice expressing the BDC2.5 TCR transgene are useful for studying type 1 diabetes. Several peptides have been identified that are highly active in stimulating BDC2.5 T cells. Herein, we describe the use of I-Ag7 tetramers containing two such peptides, p79 and p17, to detect and characterize peptide-specific T cells. The tetramers could stain CD4+ T cells in the islets and spleens of BDC2.5 transgenic mice. The percentage of CD4+, tetramer+ T cells increased in older mice, and it was generally higher in the islets than in the spleens. Our results also showed that tetAg7/p79 could stain a small population of CD4+ T cells in both islets and spleens of NOD mice. The percentage of CD4+, tetramer+ T cells increased in cells that underwent further cell division after being activated by peptides. The avidity of TCRs on purified tetAg7/p79+ T cells for tetAg7/p79 was slightly lower than that of BDC2.5 T cells. Although tetAg7/p79+ T cells, like BDC2.5 T cells, secreted a large quantity of IFN-γ, they were biased toward being IL-10-producing cells. Additionally, <3% of these cells expressed TCR Vβ4. In vivo adoptive transfer experiments showed that NOD/scid recipient mice cotransferred with tetAg7/p79+ T cells and NOD spleen cells, like mice transferred with NOD spleen cells only, developed diabetes. Therefore, we have generated Ag-specific tetramers that could detect a heterogeneous population of T cells, and a very small number of NOD mouse T cells may represent BDC2.5-like cells.
Optimal Chemotactic Responses of Leukemic T Cells to Stromal Cell-Derived Factor-1 Requires the Activation of Both Class IA and IB Phosphoinositide 3-Kinases
Authors: Curnock Adam P. , Sotsios Yannis , Wright Karen L. , Ward Stephen G.
Published: April 2014

Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 are a multifunctional chemokine/receptor system with essential roles in the development of the immune system and other aspects of embryogenesis, including vascularization and organ development. SDF-1 is also a potent chemoattractant for T cells and has roles in both inflammation and immune homeostasis. Our group has previously demonstrated that phosphoinositide 3-kinase (PI 3-kinase) is activated in SDF-1-stimulated T cells and is indeed required for SDF-1-mediated chemotaxis. In this study Jurkat clones were established, stably expressing dominant negative constructs of class IA and class IB PI 3-kinases under the control of the tetracycline off inducible gene system, to determine the relative roles of these PI 3-kinases in SDF-1 signaling. Our results show that expression of either kinase-dead PI3Kγ (KD-PI3Kγ) or Δp85 (a construct unable to bind class IA p110α, -β, or -δ) leads to a partial inhibition of SDF-1-stimulated protein kinase B phosphorylation, but had no effect on SDF-1-induced phosphorylation of the mitogen-activated protein kinase ERK1/2. Functional studies demonstrated that expression of KD-PI3Kγ markedly inhibited SDF-1-mediated chemotaxis, typically eliciting 40–60% inhibition. Interestingly, the expression of Δp85 also leads to inhibition of the SDF-1-mediated chemotactic response, albeit to a much lesser extent than achieved with the KD-PI3Kγ mutant, typically in the range of 20–40% inhibition. Furthermore, the inhibition of chemotaxis by the expression of dominant negative class IA or class IB PI 3-kinases could be enhanced by the presence of the PI 3-kinase inhibitor LY294002. Together, these results demonstrate that optimal chemotactic response of leukemic T cells to SDF-1 requires the activation of both class IA and class IB PI 3-kinases.
CpG-A-Induced Monocyte IFN-γ-Inducible Protein-10 Production Is Regulated by Plasmacytoid Dendritic Cell-Derived IFN-α
Authors: Blackwell Sue E. , Krieg Arthur M.
Published: April 2014

Unmethylated CpG motifs in bacterial DNA or synthetic oligodeoxynucleotides (ODN) are known for inducing a Th1 cytokine/chemokine environment, but the mechanisms regulating this have been unclear. Recent studies have defined two classes of CpG ODN, CpG-A ODN that induce plasmacytoid dendritic cells (pDC) to secrete very high levels of IFN-α, and CpG-B ODN that induce only low levels of IFN-α production, but strongly activate B cells. We now demonstrate that a CpG-A ODN directly activates pDC secretion of IFN-α and other soluble factors that secondarily induce purified monocytes to secrete high levels of the Th1-promoting chemokine IFN-γ-inducible protein-10 (IP-10). Cell contact between the monocytes and pDC is not required for this interaction. IFN-α is necessary, but only partially sufficient, for this indirect CpG-induced monocyte IP-10 production. Although CpG ODN induce human PBMC to make only very slight amounts of IFN-γ, we find that these low concentrations synergize with IFN-α for inducing monocyte production of IP-10. These studies provide a better understanding of the mechanisms through which CpG ODN create a Th1-like environment.
Mature Dendritic Cells Derived from Human Monocytes Within 48 Hours: A Novel Strategy for Dendritic Cell Differentiation from Blood Precursors
Authors: Dauer Marc , Obermaier Bianca , Herten Jan , Haerle Carola , Pohl Katrin , Rothenfusser Simon , Schnurr Max , Endres Stefan , Eigler Andreas
Published: April 2014

It is widely believed that generation of mature dendritic cells (DCs) with full T cell stimulatory capacity from human monocytes in vitro requires 5–7 days of differentiation with GM-CSF and IL-4, followed by 2–3 days of activation. Here, we report a new strategy for differentiation and maturation of monocyte-derived DCs within only 48 h of in vitro culture. Monocytes acquire immature DC characteristics by day 2 of culture with GM-CSF and IL-4; they down-regulate CD14, increase dextran uptake, and respond to the inflammatory chemokine macrophage inflammatory protein-1α. To accelerate DC development and maturation, monocytes were incubated for 24 h with GM-CSF and IL-4, followed by activation with proinflammatory mediators for another 24 h (FastDC). FastDC expressed mature DC surface markers as well as chemokine receptor 7 and secreted IL-12 (p70) upon CD40 ligation in the presence of IFN-γ. The increase in intracellular calcium in response to 6Ckine showed that chemokine receptor 7 expression was functional. When FastDC were compared with mature monocyte-derived DCs generated by a standard 7-day protocol, they were equally potent in inducing Ag-specific T cell proliferation and IFN-γ production as well as in priming autologous naive T cells using tetanus toxoid as a model Ag. These findings indicate that FastDC are as effective as monocyte-derived DCs in stimulating primary, Ag-specific, Th 1-type immune responses. Generation of FastDC not only reduces labor, cost, and time required for in vitro DC development, but may also represent a model more closely resembling DC differentiation from monocytes in vivo.
Direct Visualization of Cross-Reactive Effector and Memory Allo-Specific CD8 T Cells Generated in Response to Viral Infections
Authors: Brehm Michael A. , Markees Thomas G. , Daniels Keith A. , Greiner Dale L. , Rossini Aldo A. , Welsh Raymond M.
Published: April 2014

CD8 T cell cross-reactivity between heterologous viruses has been shown to provide protective immunity, induce immunopathology, influence the immunodominance of epitope-specific T cell responses, and shape the overall memory population. Virus infections also induce cross-reactive allo-specific CTL responses. In this study, we quantified the allo-specific CD8 T cells elicited by infection of C57BL/6 (B6) mice with lymphocytic choriomeningitis virus (LCMV). Cross-reactive LCMV-specific CD8 T cells were directly visualized using LCMV peptide-charged MHC tetramers to costain T cells that were stimulated to produce intracellular IFN-γ in response to allogeneic target cells. The cross-reactivity between T cells specific for LCMV and allogeneic Ags was broad-based, in that it involved multiple LCMV-derived peptides, but there were distinctive patterns of reactivity against allogeneic cells with different haplotypes. Experiments indicated that this cross-reactivity was not due to the expression of two TCR per cell, and that the patterns of allo-reactivity changed during sequential infection with heterologous viruses. The allo-specific CD8 T cells generated by LCMV infection were maintained at relatively high frequencies in the memory pool, indicating that memory allo-specific CD8 T cell populations can arise as a consequence of viral infections. Mice previously infected with LCMV and harboring allo-specific memory T cells were refractory to the induction of tolerance to allogeneic skin grafts.
The gp49B1 Inhibitory Receptor Regulates the IFN-γ Responses of T Cells and NK Cells
Authors: Gu Xiaogang , Laouar Amale , Wan Junmei , Daheshia Massoud , Lieberman Judy , Yokoyama Wayne M. , Katz Howard R. , Manjunath N.
Published: April 2014

The magnitude and diversity of Ag-specific T cell effector activity have been proposed to be controlled by an integration of positive signals transduced by the TCR and negative signals originating from inhibitory cell surface molecules. Although the lectin family of NK cell-associated inhibitory receptors has been reported to regulate the function of murine CTLs, gp49B1, the Ig superfamily member is not known to be expressed on T cells. Moreover, the consequences of the lack of an endogenously expressed NK cell-associated inhibitory receptor on T cell functions are not known. We report that gp49B1 is expressed by nearly all activated CD8 and CD4 T cells in addition to NK cells during an immune response to viral, bacterial, or tumor challenge. Kinetics of gp49B1 expression parallel functional capability and subside in the memory phase. Following vaccinia viral infection, IFN-γ production by both subsets of T cells and NK cells is enhanced in gp49B1-deficient mice compared with gp49B1+/+ mice. The stimulation threshold for IFN-γ production is also lower in gp49B1-deficient T cells. In contrast, no significant differences were observed in the cytotoxic responses. We conclude that gp49B1 is a unique inhibitory receptor that is induced in multiple lineages of innate and adaptive immune cells during an infection and controls their IFN-γ, but not cytotoxic responses.
A Subset of Toll-Like Receptor Ligands Induces Cross-presentation by Bone Marrow-Derived Dendritic Cells
Authors: Datta Sandip K. , Redecke Vanessa , Prilliman Kiley R. , Takabayashi Kenji , Corr Maripat , Tallant Thomas , DiDonato Joseph , Dziarski Roman , Akira Shizuo , Schoenberger Stephen P. , Raz Eyal
Published: April 2014

Dendritic cells (DCs) are capable of cross-presenting exogenous Ag to CD8+ CTLs. Detection of microbial products by Toll-like receptors (TLRs) leads to activation of DCs and subsequent orchestration of an adaptive immune response. We hypothesized that microbial TLR ligands could activate DCs to cross-present Ag to CTLs. Using DCs and CTLs in an in vitro cross-presentation system, we show that a subset of microbial TLR ligands, namely ligands of TLR3 (poly(inosinic-cytidylic) acid) and TLR9 (immunostimulatory CpG DNA), induces cross-presentation. In contrast to presentation of Ag to CD4+ T cells by immature DCs, TLR-induced cross-presentation is mediated by mature DCs, is independent of endosomal acidification, and relies on cytosolic Ag processing machinery.
APCs Present Aβk-Derived Peptides That Are Autoantigenic to Type B T Cells
Authors: Lovitch Scott B. , Walters James J. , Gross Michael L. , Unanue Emil R.
Published: April 2014

Type B T cells recognize peptide provided exogenously but are ignorant of the same epitope derived from intracellular processing. In this study, we demonstrate the existence of type B T cells to an abundant autologous peptide derived from processing of the I-Ak β-chain. T cell hybridomas raised against this peptide fail to recognize syngeneic APC despite abundant presentation of the naturally processed epitope but react in a dose-dependent manner to exogenous peptide. Moreover, these hybridomas respond to Aβk peptide extracted from the surface of I-Ak-expressing APC. This peptide was isolated from B cell lines where it was found in high abundance; it was also present in lines lacking HLA-DM, but in considerably lower amounts. Therefore, type B T cells exist in the naive repertoire to abundant autologous peptides. We discuss the implications of these findings to the potential biological role of type B T cells in immune responses and autoimmune pathology.
Host Absence of CCR5 Potentiates Dendritic Cell Vaccination
Authors: Ng-Cashin Judith , Kuhns Jennifer J. , Burkett Susan E. , Powderly John D. , Craven Robin R. , van Deventer Hank W. , Kirby Suzanne L. , Serody Jonathan S.
Published: April 2014

Previous work has shown that dendritic cells (DCs) express specific chemokine receptors that allow for coordinated movement in vivo. To test the in vivo relevance of this, we used a murine melanoma system and knockout mice to investigate the function of the chemokine receptor CCR5 and its ligands, CCR ligand (CCL)3 and CCL5. We found that the lack of CCR5 in the host mouse resulted in delayed tumor growth, but this effect was overcome at a higher tumor load. With the administration of tumor charged DCs, CCR5−/− mice that had previously been injected with tumor were completely protected from tumor. This effect was dependent on the dose of tumor cells and the expression of CCR5 on the DC and its absence in the host. In contrast, the loss of the CCR5 ligand, CCL3, led to an early delay in tumor growth that did not persist, while the absence of the CCR5 ligand, CCL5, had no effect. Blocking the activity of CCR5 in the host may represent a new strategy for enhancing the activity of a therapeutic melanoma DC vaccine.
Hyperoxia Mediates Acute Lung Injury and Increased Lethality in MurineLegionellaPneumonia: The Role of Apoptosis
Authors: Tateda Kazuhiro , Deng Jane C. , Moore Thomas A. , Newstead Michael W. , Paine Robert , Kobayashi Nobuyuki , Yamaguchi Keizo , Standiford Theodore J.
Published: April 2014

Legionella pneumophila is a major cause of life-threatening pneumonia, which is characterized by a high incidence of acute lung injury and resultant severe hypoxemia. Mechanical ventilation using high oxygen concentrations is often required in the treatment of patients with L. pneumophila pneumonia. Unfortunately, oxygen itself may propagate various forms of tissue damage, including acute lung injury. The effect of hyperoxia as a cofactor in the course of L. pneumophila pneumonia is poorly understood. In this study, we show that exposure to hyperoxic conditions during the evolution of pneumonia results in a marked increase in lethality in mice with Legionella pneumonia. The enhanced lethality was associated with an increase in lung permeability, but not changes in either lung bacterial burden or leukocyte accumulation. Interestingly, accelerated apoptosis as evidenced by assessment of histone-DNA fragments and caspase-3 activity were noted in the infected lungs of mice exposed to hyperoxia. TUNEL staining of infected lung sections demonstrated increased apoptosis in hyperoxic mice, predominantly in macrophages and alveolar epithelial cells. In vitro exposure of primary murine alveolar epithelial cells to Legionella in conjunction with hyperoxia accelerated apoptosis and loss of barrier function. Fas-deficient mice demonstrated partial resistance to the lethal effects of Legionella infection induced by hyperoxia, which was associated with attenuated apoptosis in the lung. These results demonstrate that hyperoxia serves as an important cofactor for the development of acute lung injury and lethality in L. pneumophila pneumonia. Exaggerated apoptosis, in part through Fas-mediated signaling, may accelerate hyperoxia-induced acute lung injury in Legionella pneumonia.
A Chimeric Multi-Human Epidermal Growth Factor Receptor-2 B Cell Epitope Peptide Vaccine Mediates Superior Antitumor Responses
Authors: Dakappagari Naveen K. , Pyles John , Parihar Robin , Carson William E. , Young Donn C. , Kaumaya Pravin T. P.
Published: April 2014

Immunotherapeutic approaches to cancer should focus on novel undertakings that modulate immune responses by synergistic enhancement of antitumor immunological parameters. Cancer vaccines should preferably be composed of multiple defined tumor Ag-specific B and T cell epitopes. To develop a multiepitope vaccine, 12 high ranking B cell epitopes were identified from the extracellular domain of the human epidermal growth factor receptor-2 (HER-2) oncoprotein by computer-aided analysis. Four novel HER-2 B cell epitopes were synthesized as chimeras with a promiscuous T cell epitope (aa 288–302) from the measles virus fusion protein (MVF). Two chimeric peptide vaccines, MVF HER-2316–339 and MVF HER-2485–503 induced high levels of Abs in outbred rabbits, which inhibited tumor cell growth. In addition, Abs induced by a combination of two vaccines, MVF HER-2316–339 and MVF HER-2628–647 down-modulated receptor expression and activated IFN-γ release better than the individual vaccines. Furthermore, this multiepitope vaccine in combination with IL-12 caused a significant reduction (p = 0.004) in the number of pulmonary metastases induced by challenge with syngeneic tumor cells overexpressing HER-2. Peptide Abs targeting specific sites in the extracellular domain may be used for exploring the oncoprotein’s functions. The multiepitope vaccine may have potential application in the treatment of HER-2-associated cancers.
TCR Vβ8+ T Cells Prevent Development of Toxoplasmic Encephalitis in BALB/c Mice Genetically Resistant to the Disease
Authors: Kang Hoil , Liesenfeld Oliver , Remington Jack S. , Claflin Jennifer , Wang Xisheng , Suzuki Yasuhiro
Published: April 2014

BALB/c are genetically resistant to development of toxoplasmic encephalitis (TE) when infected with Toxoplasma gondii, whereas CBA/Ca mice are susceptible. We compared TCR Vβ chain usage in lymphocytes infiltrated into brains between these animals following infection. TCR Vβ8+ cells were the most frequent T cell population in brains of infected, resistant BALB/c mice, whereas TCR Vβ6+ T cells were more prevalent than Vβ8+ T cells in brains of infected, susceptible CBA/Ca mice. Adoptive transfer of Vβ8+ immune T cells, obtained from infected BALB/c mice, prevented development of TE and mortality in infected athymic nude mice that lack T cells. In contrast, adoptive transfer of Vβ6+ immune T cells did not prevent development of TE or mortality in the nude mice. The protective activity of Vβ8+ immune T cells was greater than that of the total Vβ8− population. In addition, Vβ8+ immune T cells produced markedly greater amounts of IFN-γ than did the Vβ8− population after stimulation with tachyzoite lysate Ags in vitro. Thus, Vβ8+ T cells appear to play a crucial role in the genetic resistance of BALB/c mice against development of TE.
Identification and Characterization of the Immunodominant Rat HER-2/neu MHC Class I Epitope Presented by Spontaneous Mammary Tumors from HER-2/neu-Transgenic Mice
Authors: Ercolini Anne M. , Machiels Jean-Pascal H. , Chen Yi Cheng , Slansky Jill E. , Giedlen Martin , Reilly R. Todd , Jaffee Elizabeth M.
Published: April 2014

The HER-2/neu (neu-N)-transgenic mice are a clinically relevant model of breast cancer. They are derived from the parental FVB/N mouse strain and are transgenic for the rat form of the proto-oncogene HER-2/neu (neu). In this study, we report the identification of a MHC class I peptide in the neu protein that is recognized by CD8+ T cells derived from vaccinated FVB/N mice. This 10-mer was recognized by all tumor-specific FVB/N T cells generated regardless of the TCR Vβ region expressed by the T cell or the method of vaccination used, establishing it as the immunodominant MHC class I epitope in neu. T cells specific for this epitope were able to cure FVB/N mice of transplanted neu-expressing tumor cells, demonstrating that this is a naturally processed peptide. Altered peptide analogs of the epitope were analyzed for immunogenicity. Vaccination with dendritic cells pulsed with a heteroclitic peptide provided FVB/N and neu-N mice with increased protection against tumor challenge as compared with mice immunized with dendritic cells loaded with either wild-type or irrelevant peptide. Discovery of this epitope allows for better characterization of the CD8+ T cell responses in the neu-N mouse model in which neu-specific tolerance must be overcome to produce effective antitumor immunity.
A Replication Competent Adenovirus 5 Host Range Mutant-Simian Immunodeficiency Virus (SIV) Recombinant Priming/Subunit Protein Boosting Vaccine Regimen Induces Broad, Persistent SIV-Specific Cellular Immunity to Dominant and Subdominant Epitopes in Mamu-A*01 Rhesus Macaques
Authors: Malkevitch Nina , Patterson L. Jean , Aldrich Kristine , Richardson Ersell , Alvord W. Gregory , Robert-Guroff Marjorie
Published: April 2014

CTL are important in controlling HIV and SIV infection. To quantify cellular immune responses induced by immunization, CD8+ T cells specific for the subdominant Env p15m and p54m epitopes and/or the dominant Gag p11C epitope were evaluated by tetramer staining in nine macaques immunized with an adenovirus (Ad) 5 host range mutant (Ad5hr)-SIVenv/rev recombinant and in four of nine which also received an Ad5hr-SIVgag recombinant. Two Ad5hr-SIV recombinant priming immunizations were followed by two boosts with gp120 protein or an envelope polypeptide representing the CD4 binding domain. Two mock-immunized macaques served as controls. IFN-γ-secreting cells were also assessed by ELISPOT assay using p11C, p15m, and p54m peptide stimuli and overlapping pooled Gag and Env peptides. As shown by tetramer staining, Ad-recombinant priming elicited a high frequency of persistent CD8+ T cells able to recognize p11C, p15m, and p54m epitopes. The presence of memory cells 38 wk postinitial immunization was confirmed by expansion of tetramer-positive CD8+ T cells following in vitro stimulation. The SIV-specific CD8+ T cells elicited were functional and secreted IFN-γ in response to SIV peptide stimuli. Although the level and frequency of response of peripheral blood CD8+ T cells to the subdominant Env epitopes were not as great as those to the dominant p11C epitope, elevated responses were observed when lymph node CD8+ T cells were evaluated. Our data confirm the potency and persistence of functional cellular immune responses elicited by replication competent Ad-recombinant priming. The cellular immunity elicited is broad and extends to subdominant epitopes.
Up-Regulation of Cysteinyl Leukotriene 1 Receptor by IL-13 Enables Human Lung Fibroblasts to Respond to Leukotriene C4 and Produce Eotaxin
Authors: Chibana Kazuyuki , Ishii Yoshiki , Asakura Takuma , Fukuda Takeshi
Published: April 2014

Cysteinyl leukotrienes (CysLTs) play an important role in eosinophilic airway inflammation. In addition to their direct chemotactic effects on eosinophils, indirect effects have been reported. Eotaxin is a potent eosinophil-specific chemotactic factor produced mainly by fibroblasts. We investigated whether CysLTs augment eosinophilic inflammation via eotaxin production by fibroblasts. Leukotriene (LT)C4 alone had no effect on eotaxin production by human fetal lung fibroblasts (HFL-1). However, LTC4 stimulated eotaxin production by IL-13-treated fibroblasts, thereby indirectly inducing eosinophil sequestration. Unstimulated fibroblasts did not respond to LTC4, but coincubation or preincubation of fibroblasts with IL-13 altered the response to LTC4. To examine the mechanism(s) involved, the expression of CysLT1R in HFL-1 was investigated by quantitative real-time PCR and flow cytometry. Only low levels of CysLT1R mRNA and no CysLT1R protein were expressed in unstimulated HFL-1. In contrast, stimulation with IL-13 at a concentration of 10 ng/ml for 24 h significantly up-regulated both CysLT1R mRNA and protein expression in HFL-1. The synergistic effect of LTC4 and IL-13 on eotaxin production was abolished by CysLT1R antagonists pranlukast and montelukast. These findings suggest that IL-13 up-regulates CysLT1R expression, which may contribute to the synergistic effect of LTC4 and IL-13 on eotaxin production by lung fibroblasts. In the Th2 cytokine-rich milieu, such as that in bronchial asthma, CysLT1R expression on fibroblasts might be up-regulated, thereby allowing CysLTs to act effectively and increase eosinophilic inflammation.
Frequencies of Circulating Cytolytic, CD45RA+CD27−, CD8+ T Lymphocytes Depend on Infection with CMV
Authors: Kuijpers Taco W. , Vossen Mireille T. , Gent Mi-Ran , Davin Jean-Claude , Roos Marijke T. , Wertheim-van Dillen Pauline M. , Weel Jan F. , Baars Paul A. , van Lier Rene A.
Published: April 2014

Viral infections may cause serious disease unless the adaptive immune system is able to clear the viral agents through its effector arms. Recent identification and functional characterization of subpopulations of human CD8+ T cells has set the stage to study the correlation between the appearance of particular subsets and common viral infections during childhood, i.e., EBV, CMV, varicella-zoster virus (VZV), and the attenuated measles-mumps-rubella (MMR) vaccine strains. In a cohort of 220 healthy children we analyzed lymphocytes and subpopulations of CD4+ and CD8+ T cells. The presence of the cytolytic CD45RA+CD27− subset of CD8+ T cells correlated with prior CMV infection as defined by seroconversion (p < 0.0001). The number of this CD8+ T cell subset remained stable during follow-up over 3 years in 40 children. The CD45RA+CD27− subset of CD8+ T cells first appeared during acute CMV infection and subsequently stabilized at an individual set-point defined by age and immunocompetence. The functional importance of these cells in CMV surveillance was reflected by their increased numbers in immunosuppressed pediatric kidney transplant patients. Preferential expansion of CD8+CD45RA+CD27− cytolytic T cells seems unique for CMV.
Cutting Edge: Increased NK Cell Activity in HIV-1-Exposed but Uninfected Vietnamese Intravascular Drug Users
Authors: Scott-Algara Daniel , Truong Lien X. , Versmisse Pierre , David Annie , Luong Tram T. , Nguyen Ngai V. , Theodorou Ioannis , Barré-Sinoussi Françoise , Pancino Gianfranco
Published: April 2014

We addressed the role of innate immunity in the protection against HIV-1 infection by studying NK cell function in 37 Vietnamese intravascular drug users (IDUs), who appeared to remain HIV-1 uninfected despite many years of high-risk exposure (exposed uninfected, EU), 10 IDUs who underwent seroconversion and 28 unexposed blood donors. Main results were: NK cell lytic activities against both the NK-susceptible K562 cell line and the NK-resistant Daudi cell line were significantly augmented in EU IDUs compared with either controls or seroconverters before or after seroconversion; NK cells producing the cytokines IFN-γ and TNF-α and the β chemokines CCL3, CCL4, and CCL5 were also increased in the EU IDUs, either after in vitro activation or without stimulation. The finding of an enhanced NK cell function in EU IDUs, especially compared with IDUs who became HIV-1 infected, supports the hypothesis that NK cells contribute to the protection against HIV-1 infection.
Cutting Edge: TCR Contacts as Anchors: Effects on Affinity and HLA-DM Stability
Authors: Anderson Matthew W. , Gorski Jack
Published: April 2014

Peptides presented via the class II MHC (MHCII) pathway are selected based on affinity for MHCII and stability in the presence of HLA-DM. Currently, epitope selection is thought to be controlled by the ability of peptide to sequester “anchor” residues into pockets in the MHCII. Residues exhibiting higher levels of solvent accessibility have been shown to contact TCR, but their roles in affinity and complex stability have not been directly studied. Using the HLA-DR1-binding influenza peptide, hemagglutinin (306–318), as a model, we show that side chain substitutions at these positions influence affinity and HLA-DM stability. Multiple substitutions reduce affinity to a greater extent than the loss of the major P1 anchor residue. We propose that these effects may be mediated through the H-bond network. These results demonstrate the importance of solvent-exposed residues in epitope selection and blur the distinctions between anchor and TCR contact residues.
Requirements for T Cell-Polarized Tubulation of Class II+ Compartments in Dendritic Cells
Authors: Bertho Nicolas , Cerny Jan , Kim You-Me , Fiebiger Edda , Ploegh Hidde , Boes Marianne
Published: April 2014

Activation of naive CD4 T cells by dendritic cells requires the sequential interaction of many TCR molecules with peptide-class II complexes of the appropriate specificity. Such interaction results in morphological transformation of class II MHC-containing endosomal compartments. In this study, we analyze the requirements for long tubular endosomal structures that polarize toward T cell contact sites using dendritic cells from I-Ab class II -enhanced green fluorescent protein knock-in mice and I-Ab-restricted CD4 T cells specific for OVA. Clustering of membrane proteins and ligation of T cell adhesion molecules LFA-1 and CD2 are involved in induction of endosomal tubulation. Activation of T cells increases their ability to induce class II-enhanced green fluorescent protein-positive tubules in dendritic cells, in part through up-regulation of CD40 ligand. Remarkably, and in stark contrast with the result obtained with dendritic cells loaded with intact OVA, OVA peptide added to dendritic cells failed to evoke T cell-polarized endosomal tubulation even though both conditions allowed T cell stimulation. These results suggest the existence of microdomains on the membrane of dendritic cells that allow Ag-specific T cells to evoke tubulation in the dendritic cell.
IL-2 Is Not Required for the Initiation of CD8 T Cell Cycling but Sustains Expansion
Authors: D’Souza Warren N. , Lefrançois Leo
Published: April 2014

Based primarily on in vitro data, IL-2 is believed to be the key cytokine for initiation of the cell cycle of activated T cells. However, the role of IL-2 remains unresolved for T cell responses in vivo. We examined whether the absence of IL-2-mediated signaling in CD8 T cells affected initiation of proliferation. Our results conclusively demonstrated that initial division of Ag-specific CD8 T cells following priming was IL-2 independent, regardless of the context in which Ag was presented. In contrast, the latter stage of the proliferative phase was IL-2-dependent, particularly in nonlymphoid tissues. Thus, activated CD8 T cells initially undergo IL-2-independent proliferation, but reach a critical juncture where the requirement for IL-2 as a growth factor gains prominence.
Dendritic Cell-Induced Activation of Adaptive and Innate Antitumor Immunity
Authors: van den Broeke Leon T. , Daschbach Emily , Thomas Elaine K. , Andringa Gerda , Berzofsky Jay A.
Published: April 2014

While studying Ag-pulsed syngeneic dendritic cell (DC) immunization, we discovered that surprisingly, unpulsed DCs induced protection against tumor lung metastases resulting from i.v. injection of a syngeneic BALB/c colon carcinoma CT26 or a syngeneic C57BL/6 lung carcinoma LL/2. Splenocytes or immature splenic DCs did not protect. The protection was mediated by NK cells, in that it was abrogated by treatment with anti-asialo-GM1 but not anti-CD8, and was induced by CD1−/− DCs unable to stimulate NKT cells, but did not occur in beige mice lacking NK cells. Protection correlated with increased NK activity, and increased infiltration of NK but not CD8+ cells in lungs of tumor-bearing mice. Protection depended on the presence of costimulatory molecules CD80, CD86, and CD40 on the DCs, but surprisingly did not require DCs that could make IL-12 or IL-15. Unexpectedly, protection sensitive to anti-asialo-GM1 and increased NK activity were still present 14 mo after DC injection. As NK cells lack memory, we found by depletion that CD4+ not CD8+ T cells were required for induction of the NK antitumor response. The role of DCs and CD4+ T cells provides a novel mechanism for NK cell induction and innate immunity against cancer that may have potential in preventing clinical metastases.
Ligation of CD27 on B Cells In Vivo during Primary Immunization Enhances Commitment to Memory B Cell Responses
Authors: Raman Vanitha S. , Akondy Rama S. , Rath Satyajit , Bal Vineeta , George Anna
Published: April 2014

Ligation of CD27 on B cells has been shown to inhibit terminal differentiation of activated murine B cells into plasma cells. We show in this study that this inhibition is accompanied by an enhanced movement of activated B cells toward differentiation into memory cells. Treatment of mice with anti-CD27 during immunization leads to the generation of greater numbers of Ag-binding B cells in draining lymph nodes that persist for longer periods of time, and they contain a greater proportion of cells of a postgerminal center phenotype. Limiting dilution analyses reveal that they contain a higher frequency of cells that can be stimulated to secrete specific IgG, and adoptive transfer experiments confirm that they can generate higher secondary responses in carrier-primed recipients. Remarkably, significant secondary responses are also seen following primary immunization with a T-independent Ag in the presence of anti-CD27, confirming that ligation of CD27 on B cells during priming induces differentiation into the memory lineage. Treatment with anti-CD27 during priming also increases the average affinity of the secondary response, suggesting that high affinity clones generated early in a primary response may normally differentiate preferentially into plasma cells and are rescued from this fate by CD27 ligation. Anti-CD40 treatment shows similar effects in vivo. However, unlike CD27, CD40 coligation also enhances proliferation, survival, and isotype switching of LPS-stimulated B cells, suggesting that the two receptors may enhance commitment to B cell memory by different mechanisms, or that a common mechanism is used through both receptors that does not involve cell cycle control or survival.
Tolerance through Indifference: Autoreactive B Cells to the Nuclear Antigen La Show No Evidence of Tolerance in a Transgenic Model
Authors: Aplin Brett D. , Keech Catherine L. , de Kauwe Andrea L. , Gordon Thomas P. , Cavill Dana , McCluskey James
Published: April 2014

Systemic autoimmune diseases are characterized by the production of high titer autoantibodies specific for ubiquitous nuclear self-Ags such as DNA, Sm, and La (SS-B), so the normal mechanisms of B cell tolerance to disease-associated nuclear Ags have been of great interest. Mechanisms of B cell tolerance include deletion, anergy, developmental arrest, receptor editing, and B cell differentiation to the B-1 subtype. However, recent studies in our laboratory have suggested that B cell tolerance to the nuclear autoantigen La is limited in normal mice, and tolerance may reside primarily in the T cell compartment. To test this hypothesis, we created Ig transgenic mice expressing the IgM H chain from an mAb specific for a xenogeneic epitope within human La (hLa). These mice were bred with hLa-transgenic mice that constitutively express hLa in a manner comparable to endogenous mouse La. Between 5–15% of transgenic B cells developing in the absence of hLa were specific for hLa, and these cells were neither depleted nor developmentally arrested in the presence of endogenous hLa expression. Instead, these autoreactive B cells matured normally and differentiated into Ab-forming cells, capable of secreting high titer autoantibody. Additionally, the life span of autoreactive hLa-specific B cells was not reduced, and they were phenotypically and functionally indistinguishable from naive nonautoreactive hLa-specific B cells developing in the absence of hLa. Together these data suggest a lack of intrinsic B cell tolerance involving any known mechanisms indicating that these autoreactive B cells are indifferent to their autoantigen.
Vaccination with Plasmid DNA Activates Dendritic Cells via Toll-Like Receptor 9 (TLR9) but Functions in TLR9-Deficient Mice
Authors: Spies Barbara , Hochrein Hubertus , Vabulas Martin , Huster Katharina , Busch Dirk H. , Schmitz Frank , Heit Antje , Wagner Hermann
Published: April 2014

We analyzed whether the immunobiology of vaccinating plasmid DNA containing a transcription unit for OVA is influenced by immunostimulatory CpG motifs in the plasmid backbone. Indeed, plasmid DNA differentially activated in vitro myeloid and plasmacytoid dendritic cells (DCs) provided they expressed the CpG-DNA receptor, Toll-like receptor 9 (TLR9). Dependent on the DC subset, activation resulted in type 1 IFN production, while both DC subsets produced IL-6 and up-regulated expression of costimulatory molecules CD40 and CD86. In vivo, however, even upon repeated vaccination with plasmid DNA, priming of OVA-specific CTL and clonal expansion of SIINFEKL-specific CD8 T cells were equal in TLR9-positive and TLR9- or MyD88-negative mice. Overall, these results negate a dominant role of CpG-DNA/TLR9 interactions in long-term vaccination protocols.
CD19 Function in Early and Late B Cell Development. II. CD19 Facilitates the Pro-B/Pre-B Transition
Authors: Otero Dennis C. , Rickert Robert C.
Published: April 2014

Proliferative expansion of pro-B cells is an IL-7-dependent process that allows for the rearrangement of H chain genes and the expression of the pre-B cell receptor (pre-BCR). Further B cell differentiation is dependent upon signals elicited through the pre-BCR, which are thought to be responsible for allelic exclusion, induced L chain gene rearrangement, and continued proliferation. CD19 promotes the proliferation and survival of mature B cells, but its role in early B cell development is less well understood. Here we identify and characterize impairments in early B cell development in CD19−/− mice. Following sublethal irradiation, we found decreased numbers of autoreconstituted early B cells, which was first evident in the large cycling pre-B cell fraction. Reduced cell progression due to a defect in proliferation was made evident from cell cycle analysis and bromodeoxyuridine labeling of bone marrow cells from CD19−/− and wild-type mice. Studies of IL-7-dependent pre-B cell cultures derived from wild-type and CD19−/− mouse bone marrow suggested that CD19 has little affect on IL-7 signaling. By contrast, signaling through the pre-BCR was impaired in the absence of CD19, as demonstrated by reduced activation of Bruton’s tyrosine kinase and extracellular signal-regulated kinase/mitogen-activated protein kinase. Thus, in addition to promoting mature B cell homeostasis and Ag-induced responses, the early onset of CD19 expression acts to enhance B cell generation.
Dendritic Cells Charged with Apoptotic Tumor Cells Induce Long-Lived Protective CD4+ and CD8+ T Cell Immunity against B16 Melanoma
Authors: Goldszmid Romina S. , Idoyaga Juliana , Bravo Alicia I. , Steinman Ralph , Mordoh José , Wainstok Rosa
Published: April 2014

Dendritic cells (DCs) are potent APCs and attractive vectors for cancer immunotherapy. Using the B16 melanoma, a poorly immunogenic experimental tumor that expresses low levels of MHC class I products, we investigated whether DCs loaded ex vivo with apoptotic tumor cells could elicit combined CD4+ and CD8+ T cell dependent, long term immunity following injection into mice. The bone marrow-derived DCs underwent maturation during overnight coculture with apoptotic melanoma cells. Following injection, DCs migrated to the draining lymph nodes comparably to control DCs at a level corresponding to ∼0.5% of the injected inoculum. Mice vaccinated with tumor-loaded DCs were protected against an intracutaneous challenge with B16, with 80% of the mice remaining tumor-free 12 wk after challenge. CD4+ and CD8+ T cells were efficiently primed in vaccinated animals, as evidenced by IFN-γ secretion after in vitro stimulation with DCs loaded with apoptotic B16 or DCs pulsed with the naturally expressed melanoma Ag, tyrosinase-related protein 2. In addition, B16 melanoma cells were recognized by immune CD8+ T cells in vitro, and cytolytic activity against tyrosinase-related protein 2180–188-pulsed target cells was observed in vivo. When either CD4+ or CD8+ T cells were depleted at the time of challenge, the protection was completely abrogated. Mice receiving a tumor challenge 10 wk after vaccination were also protected, consistent with the induction of tumor-specific memory. Therefore, DCs loaded with cells undergoing apoptotic death can prime melanoma-specific helper and CTLs and provide long term protection against a poorly immunogenic tumor in mice.
Altered Regulation of FcγRII on Aged Follicular Dendritic Cells Correlates with Immunoreceptor Tyrosine-Based Inhibition Motif Signaling in B Cells and Reduced Germinal Center Formation
Authors: Aydar Yüksel , Balogh Péter , Tew John G. , Szakal Andras K.
Published: April 2014

Aging is associated with reduced trapping of Ag in the form of in immune complexes (ICs) by follicular dendritic cells (FDCs). We postulated that this defect was due to altered regulation of IC trapping receptors. The level of FDC-M1, complement receptors 1 and 2, FcγRII, and FDC-M2 on FDCs was immunohistochemically quantitated in draining lymph nodes of actively immunized mice for 10 days after Ag challenge. Initially, FDC FcγRII levels were similar but by day 3 a drastic reduction in FDC-FcγRII expression was apparent in old mice. FDC-M2 labeling, reflecting IC trapping, was also reduced and correlated with a dramatic reduction in germinal center (GC) B cells as indicated by reduced GC size and number. Nevertheless, labeling of FDC reticula with FDC-M1 and anti-complement receptors 1 and 2 was preserved, indicating that FDCs were present. FDCs in active GCs normally express high levels of FcRs that are thought to bind Fc portions of Abs in ICs and minimize their binding to FcRs on B cells. Thus, cross-linking of B cell receptor and FcR via IC is minimized, thereby reducing signaling via the immunoreceptor tyrosine-based inhibition motif. Old FDCs taken at day 3, when they lack FcγRII, were incapable of preventing immunoreceptor tyrosine-based inhibition motif signaling in wild-type B cells but old FDCs stimulated B cells from FcγRIIB−/− mice to produce near normal levels of specific Ab. The present data support the concept that FcR are regulated abnormally on old FDCs. This abnormality correlates with a reduced IC retention and with a reduced capacity of FDCs to present ICs in a way that will activate GC B cells.
Ikaros Family Members from the Agnathan Myxine glutinosa and the Urochordate Oikopleura dioica: Emergence of an Essential Transcription Factor for Adaptive Immunity
Authors: Cupit Pauline M. , Hansen John D. , McCarty Aaron S. , White Greg , Chioda Mariacristina , Spada Fabio , Smale Stephen T. , Cunningham Charles
Published: April 2014

The Ikaros multigene family encodes a number of zinc finger transcription factors that play key roles in vertebrate hemopoietic stem cell differentiation and the generation of B, T, and NK cell lineages. In this study, we describe the identification and characterization of an Ikaros family-like (IFL) protein from the agnathan hagfish Myxine glutinosa and the marine urochordate Oikopleura dioica, both of which lie on the evolutionary boundary between the vertebrates and invertebrates. The IFL molecules identified in these animals displayed high conservation in the zinc finger motifs critical for DNA binding and dimerization in comparison with those of jawed vertebrates. Expression of the IFL gene in hagfish was strongest in blood, intestine, and gills. In O. dioica, transcription from the IFL gene was initiated at or around the time of hatching and maintained throughout the life span of the animal. In situ hybridization localized O. dioica IFL expression to the Fol cells, which are responsible for generating the food filter of the house. Biochemical analysis of the DNA binding and dimerization domains from M. glutinosa and O. dioici IFLs showed that M. glutinosa behaves as a true Ikaros family member. Taken together, these results indicate that the properties associated with the Ikaros family preceded the emergence of the jawed vertebrates and thus adaptive immunity.