bluuebunny/crossref_metadata_2025 huggingface dataset:
IFN-γ-Dependent and -Independent Initiation of Switch Recombination by NK Cells
Authors: Gao Ning , Dang Tam , Yuan Dorothy
Published: April 2014

We have examined the effect of IL-2-propagated NK or NK-T cells on each of the steps required for B cell switch recombination leading to IgG2a production. The results indicate that NK cells, on their own and in the absence of IFN-γ, can induce germline transcription in resting, IgG− B lymphocytes from the γ2a locus as well as mRNA for activation-induced cytidine deaminase (AID) via a process that requires cell-cell interactions. The results also show that, in contrast to induction by T cells, activation by NK cells does not involve CD40-CD40 ligand interactions and does not extend to the induction of Iγ1 transcription. Furthermore, in contrast to stimulation by LPS and IFN-γ or by T cells, the activation events initiated by NK cells do not result in significant synthesis of functional γ2a mRNA in resting B lymphocytes even in the presence of IFN-γ. Thus, induction of germline and AID transcripts are necessary but not sufficient events for functional switching to IgG2a. These experiments, showing that NK cells themselves cannot induce IgG2a production but can polyclonally program B lymphocytes so that they preferentially switch to this isotype may explain how activated NK cells can skew the Ag-specific immune response toward IgG2a. The findings also provide further demonstration of the definitive yet limited extent of how a non-Ag-specific component of the innate system can modulate the direction of the adaptive immune response.
Combinatorial Peptide Libraries and Biometric Score Matrices Permit the Quantitative Analysis of Specific and Degenerate Interactions Between Clonotypic TCR and MHC Peptide Ligands
Authors: Zhao Yingdong , Gran Bruno , Pinilla Clemencia , Markovic-Plese Silva , Hemmer Bernhard , Tzou Abraham , Whitney Laurie Ward , Biddison William E. , Martin Roland , Simon Richard
Published: April 2014

The interaction of TCRs with MHC peptide ligands can be highly flexible, so that many different peptides are recognized by the same TCR in the context of a single restriction element. We provide a quantitative description of such interactions, which allows the identification of T cell epitopes and molecular mimics. The response of T cell clones to positional scanning synthetic combinatorial libraries is analyzed with a mathematical approach that is based on a model of independent contribution of individual amino acids to peptide Ag recognition. This biometric analysis compares the information derived from these libraries composed of trillions of decapeptides with all the millions of decapeptides contained in a protein database to rank and predict the most stimulatory peptides for a given T cell clone. We demonstrate the predictive power of the novel strategy and show that, together with gene expression profiling by cDNA microarrays, it leads to the identification of novel candidate autoantigens in the inflammatory autoimmune disease, multiple sclerosis.
Expression of TNF-α by Herpes Simplex Virus-Infected Macrophages Is Regulated by a Dual Mechanism: Transcriptional Regulation by NF-κB and Activating Transcription Factor 2/Jun and Translational Regulation Through the AU-Rich Region of the 3′ Untranslated Region
Authors: Paludan Søren R. , Ellermann-Eriksen Svend , Kruys Veronique , Mogensen Søren C.
Published: April 2014

Here we have investigated the regulation of TNF-α expression in macrophages during HSV-2 infection. Despite a low basal level of TNF-α mRNA present in resting macrophages, no TNF-α protein is detectable. HSV-2 infection marginally increases the level of TNF-α mRNA and protein in resting macrophages, whereas a strong increase is observed in IFN-γ-activated cells infected with the virus. By reporter gene assay it was found that HSV infection augments TNF-α promoter activity. Moreover, treatment of the cells with actinomycin D, which totally blocked mRNA synthesis, only partially prevented accumulation of TNF-α protein, indicating that the infection lifts a block on translation of TNF-α mRNA. EMSA analysis showed that specific binding to the κB#3 site of the murine TNF-α promoter was induced within 1 h after infection and persisted beyond 5 h where TNF-α expression is down-modulated. Binding to the cAMP responsive element site was also induced but more transiently with kinetics closely following activation of the TNF-α promoter. Inhibitors against either NF-κB activation or the activating transcription factor 2 kinase p38 abrogated TNF-α expression, showing a requirement for both signals for activation of the promoter. This observation was corroborated by reporter gene assays. As to the translational regulation of TNF-α, the AU-rich sequence in the 3′ untranslated region of the mRNA was found to be responsible for this control because deletion of this region renders mRNA constitutively translationable. These results show that TNF-α production is induced by HSV-2 in macrophages through both transcriptional and translational regulation.
Dynamic Antigen Presentation Patterns of Listeria monocytogenes-Derived CD8 T Cell Epitopes In Vivo
Authors: Škoberne Mojca , Holtappels Rafaela , Hof Herbert , Geginat Gernot
Published: April 2014

Little information exists regarding the presentation of antigenic peptides in infected tissues. In this study the in vivo presentation of four different CD8 T cell epitopes of Listeria monocytogenes was monitored. Peptide presentation was measured by a new, highly sensitive, ex vivo Ag presentation assay that was based on the testing of freshly isolated cells from infected spleens with peptide-specific CD8 T cell lines in an IFN-γ-specific ELISPOT assay. Remarkably, the peptide presentation pattern of splenocytes and that of macrophages purified from spleens of L. monocytogenes-infected mice were different from those of in vitro infected macrophage-like cell lines. The in vivo Ag presentation pattern of splenocytes also exhibited dynamic changes during the first 48 h of infection. In vivo peptide presentation at later time points postinfection was biased toward immunodominant CD8 T cell epitopes, while at an early time point, 6 h postinfection, subdominant and dominant CD8 T cell epitopes were presented with similar strength. In summary, our studies show that Ag presentation during an infection is a highly dynamic process that only can be fully appreciated by the study of cells infected in their physiological environment.
Vβ6+ T Cells Are Obligatory for Vaccine-Induced Immunity to Histoplasma capsulatum
Authors: Deepe George S. , Gibbons Reta
Published: April 2014

We examined TCR usage to a protective fragment of heat shock protein 60 from the fungus, Histoplasma capsulatum. Nearly 90% of T cell clones from C57BL/6 mice vaccinated with this protein were Vβ6+; the remainder were Vβ14+. Amino acid motifs of the CDR3 region from Vβ6+ cells were predominantly IxGGG, IGG, or SxxGG, whereas it was uniformly SFSGG for Vβ14+ clones. Short term T cell lines from Vβ6+-depleted mice failed to recognize Ag, and no T cell clones could be generated. To determine whether Vβ6+ cells were functionally important, we eliminated them during vaccination. Depletion of Vβ6+ cells abrogated protection in vivo and upon adoptive transfer of cells into TCR αβ−/− mice. Transfer of a Vβ6+, but not a Vβ14+, clone into TCR αβ−/− mice prolonged survival. Cytokine generation by Ag-stimulated splenocytes from immunized mice depleted of Vβ6+ cells was similar to that of controls. The efficacy of the Vβ6+ clone was associated with elevated production of IFN-γ, TNF-α, and GM-CSF compared with that of the Vβ14+ clone. More Vβ6+ cells were present in lungs and spleens of TCR αβ−/− on day 3 postinfection compared with Vβ14+ cells. Thus, a single Vβ family was essential for vaccine-induced immunity. Moreover, the mechanism by which Vβ6+ contributed to protective immunity differed between unfractionated splenocytes and T cell clones.
The Role of IL-4 in Heligmosomoides polygyrus-Induced Alterations in Murine Intestinal Epithelial Cell Function
Authors: Shea-Donohue Terez , Sullivan Carolyn , Finkelman Fred D. , Madden Kathleen B. , Morris Suzanne C. , Goldhill Jon , Piñeiro-Carrero Victor , Urban Joseph F.
Published: April 2014

IL-4 and IL-13 promote gastrointestinal worm expulsion, at least in part, through effects on nonlymphoid cells, such as intestinal epithelial cells. The role of IL-4/IL-13 in the regulation of intestinal epithelial function during Heligmosomoides polygyrus (Hp) infection was investigated in BALB/c mice infected with Hp or treated with a long-lasting formulation of recombinant mouse IL-4/αIL-4 complexes (IL-4C) for 7 days. Separate groups of BALB/c mice were drug-cured of initial infection and later reinfected and treated with anti-IL-4R mAb, an antagonist of IL-4 and IL-13 receptor binding, or with a control mAb. Segments of jejunum were mounted in Ussing chambers, and short circuit current responses to acetylcholine, histamine, serotonin, PGE2, and glucose were determined. Although only modest changes in epithelial cell function were observed during primary Hp infection, IL-4C or a secondary Hp infection each induced more dramatic changes, including increased mucosal permeability, reduced sodium-linked glucose absorption, and increased Cl− secretory response to PGE2. Some, but not all, effects of IL-4C and Hp infection were dependent on enteric nerves. Hp-induced changes in epithelial function were attenuated or prevented by anti-IL-4R mAb. Thus, IL-4/IL-13 mediate many of the effects of Hp infection on intestinal epithelial cell function and do so both through direct effects on epithelial cells and through indirect, enteric nerve-mediated prosecretory effects. These immune system-independent effector functions of IL-4/IL-13 may be important for host protection against gastrointestinal nematodes.
Differential Requirements for Core2 Glucosaminyltransferase for Endothelial L-Selectin Ligand Function In Vivo
Authors: Sperandio Markus , Forlow S. Bradley , Thatte Jayant , Ellies Lesley G. , Marth Jamey D. , Ley Klaus
Published: April 2014

L-selectin is a calcium-dependent lectin on leukocytes mediating leukocyte rolling in high endothelial venules and inflamed microvessels. Many selectin ligands require modification of glycoproteins by leukocyte core2 β1,6-N-acetylglucosaminyltransferase (Core2GlcNAcT-I). To test the role of Core2GlcNAcT-I for L-selectin ligand biosynthesis, we investigated leukocyte rolling in venules of untreated and TNF-α-treated cremaster muscles and in Peyer’s patch high endothelial venules (HEV) of Core2GlcNAcT-I null (core2−/−) mice. In the presence of blocking mAbs against P- and E-selectin, L-selectin-mediated leukocyte rolling was almost completely abolished in cremaster muscle venules of core2−/− mice, but not littermate control mice. By contrast, leukocyte rolling in Peyer’s patch HEV was not significantly different between core2−/− and control mice. To probe L-selectin ligands more directly, we injected L-selectin-coated beads. These beads showed no rolling in cremaster muscle venules of core2−/− mice, but significant rolling in controls. In Peyer’s patch HEV, beads coated with a low concentration of L-selectin showed reduced rolling in core2−/− mice. Beads coated with a 10-fold higher concentration of L-selectin rolled equivalently in core2−/− and control mice. Our data show that endothelial L-selectin ligands relevant for rolling in inflamed microvessels of the cremaster muscle are completely Core2GlcNAcT-I dependent. In contrast, L-selectin ligands in Peyer’s patch HEV are only marginally affected by the absence of Core2GlcNAcT-I, but are sufficiently functional to support L-selectin-dependent leukocyte rolling in Core2GlcNAcT-I-deficient mice.
Sensitized Mast Cells Migrate Toward the Agen: A Response Regulated by p38 Mitogen-Activated Protein Kinase and Rho-Associated Coiled-Coil-Forming Protein Kinase
Authors: Ishizuka Tamotsu , Okajima Fumikazu , Ishiwara Mitsuteru , Iizuka Kunihiko , Ichimonji Isao , Kawata Tadayoshi , Tsukagoshi Hideo , Dobashi Kunio , Nakazawa Tsugio , Mori Masatomo
Published: April 2014

Although mast cells accumulate within the mucosal epithelial layer of patients with allergic rhinitis and bronchial asthma, the responsible chemotactic factors are undefined. We investigated whether mast cells sensitized with Ag-specific IgE migrate toward the Ag. MC/9 mast cells sensitized with anti-DNP IgE migrated toward DNP-conjugated human serum albumin. This migration was directional, and the degree was stronger than that induced by stem cell factor. IL-3 and stem cell factor-dependent cultured mast cells derived from mouse bone marrow also migrated toward the Ag. Subsequent migration mediated by the FcεRI was significantly inhibited by incubating the cells with Y-27632, a Rho-associated coiled-coil-forming protein kinase inhibitor, or with SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor. Both p38 MAPK and MAPK-activated protein kinase (MAPKAPK)2 were activated following FcεRI aggregation, and activation of MAPKAPK2 was almost completely inhibited by 10μM SB203580. Wortmannin or a low concentration of SB203580 partially inhibited MAPKAPK2, but did not block mast cell migration. In contrast, Y-27632 did not affect the activation of MAPKAPK2. These results indicate that Ag works not only as a stimulant for allergic mediators from IgE-sensitized mast cells, but also as a chemotactic factor for mast cells. Both p38 MAPK activation and Rho-dependent activation of Rho-associated coiled-coil-forming protein kinase may be required for FcεRI-mediated cell migration.
Up-Regulation of IL-10R1 Expression Is Required to Render Human Neutrophils Fully Responsive to IL-10
Authors: Crepaldi Luca , Gasperini Sara , Lapinet José A. , Calzetti Federica , Pinardi Cristina , Liu Ying , Zurawski Sandra , de Waal Malefyt René , Moore Kevin W. , Cassatella Marco A.
Published: April 2014

We have recently shown that IL-10 fails to trigger Stat3 and Stat1 tyrosine phosphorylation in freshly isolated human neutrophils. In this study, we report that IL-10 can nonetheless induce Stat3 tyrosine phosphorylation and the binding of Stat1 and Stat3 to the IFN-γ response region or the high-affinity synthetic derivative of the c-sis-inducible element in neutrophils that have been cultured for at least 3 h with LPS. Similarly, the ability of IL-10 to up-regulate suppressor of cytokine signaling (SOCS)-3 mRNA was dramatically enhanced in cultured neutrophils and, as a result, translated into the SOCS-3 protein. Since neutrophils’ acquisition of responsiveness to IL-10 required de novo protein synthesis, we assessed whether expression of IL-10R1 or IL-10R2 was modulated in cultured neutrophils. We detected constitutive IL-10R1 mRNA and protein expression in circulating neutrophils, at levels which were much lower than those observed in autologous monocytes or lymphocytes. In contrast, IL-10R2 expression was comparable in both cell types. However, IL-10R1 (but not IL-10R2) mRNA and protein expression was substantially increased in neutrophils stimulated by LPS. The ability of IL-10 to activate Stat3 tyrosine phosphorylation and SOCS-3 synthesis and to regulate IL-1 receptor antagonist and macrophage-inflammatory protein 1β release in LPS-treated neutrophils correlated with this increased IL-10R1 expression, and was abolished by neutralizing anti-IL-10R1 and anti-IL-10R2 Abs. Our results demonstrate that the capacity of neutrophils to respond to IL-10, as assessed by Stat3 tyrosine phosphorylation, SOCS-3 expression, and modulation of cytokine production, is very dependent on the level of expression of IL-10R1.
Cutting Edge: Culture with High Doses of Viral Peptide Induces Previously Unprimed CD8+ T Cells to Produce Cytokine
Authors: Riberdy Janice M. , Zirkel Amy , Surman Sherri , Hurwitz Julia L. , Doherty Peter C.
Published: April 2014

Culturing naive T cells with 50 μM selected HIV-1 envelope peptides for 6 days in the presence of IL-2 drives the emergence of a substantial CD8+ population that secretes IFN-γ following short-term stimulation with 1 μM peptide. This response is H-2Kb restricted, epitope specific, and requires the continuing presence of peptide. The same effect was found for known H-2Db-restricted peptides from two influenza virus proteins. The great majority of these influenza-specific CD8+IFN-γ+ T cells neither stained with the cognate tetramer nor expressed the TCR Vβ bias that is characteristic of the CD8+ set expanded in vivo during an infection. Thus, multipoint binding of low affinity TCRs on naive CD8+ T cells can drive peptide-specific cytokine production. However, at least for two influenza-derived epitopes, the avidity of the TCR-MHC peptide interaction appears to be insufficient to stabilize a tetrameric complex of MHC class I glycoprotein plus peptide on the lymphocyte surface.
Cutting Edge: Dichotomy of Homing Receptor Dependence by Mucosal Effector B Cells: αE Versus L-Selectin
Authors: Csencsits Keri L. , Walters Nancy , Pascual David W.
Published: April 2014

The common mucosal immune system may be compartmentalized because lymphocyte homing to the upper respiratory tract appears to be mediated by L-selectin interactions rather than α4β7 interactions, as is the case for gut-associated lymphoreticular tissue. To assess the role of L-selectin in effector B cell immunity, L-selectin-deficient mice were intranasally immunized with cholera toxin (CT), and mucosal immune responses were compared with C57BL/6 mice. The absence of L-selectin correlated with a reduction in CT-specific secretory-IgA responses in nasal passages and reproductive tract, but not intestinal lamina propria. Cell sorting experiments showed that an L-selectin-dependent subset was responsible for CT-specific responses in nasal passages and reproductive tract, whereas an αEβ7+ B cell subset was responsible for L-selectin-independent intestinal immunity. This study provides evidence for compartmentalization of the common mucosal immune system into “intestinal” vs “nonintestinal” effector sites.
Cutting Edge: Myeloid Complement C3 Enhances the Humoral Response To Peripheral Viral Infection
Authors: Verschoor Admar , Brockman Mark A. , Knipe David M. , Carroll Michael C.
Published: April 2014

HSV-1 is the causative agent of cutaneous lesions, commonly referred to as cold sores. Primary exposure to the virus ordinarily occurs through the periphery, in particular through abraded skin or mucosal membranes. Under certain circumstances (e.g., in neonatals or AIDS patients), the infection becomes disseminated, often with severe consequences. Spread of HSV-1 is limited by virus-specific Ab. The development of an efficient humoral response to the virus is dependent on innate immunity component complement C3. The liver is the major source of C3, but there are also extrahepatic origins of C3 such as lymphoid macrophages. In the present study, the significance of C3 synthesis by bone marrow-derived cells was assessed by the transfer of wild-type bone marrow into irradiated C3-deficient mice. Using these chimeric mice, extrahepatic C3 was determined sufficient to initiate specific Ab and memory responses to a peripheral HSV-1 infection.
Cutting Edge: Amelioration of Kidney Disease in a Transgenic Mouse Model of Lupus Nephritis by Administration of the Caspase Inhibitor Carbobenzoxy-Valyl-Alanyl-Aspartyl-(β-o-methyl)-Fluoromethylketone
Authors: Seery John P. , Cattell Victoria , Watt Fiona M.
Published: April 2014

Systemic lupus erythematosus (SLE) is a common, potentially fatal, non-organ-specific autoimmune disorder. Immune complex-mediated kidney disease is the major cause of mortality. Apoptotic cells in the epidermis are a possible source of self Ags, and apoptosis of endothelial cells and lymphocytes is thought to contribute to end-organ damage. We have previously shown that female transgenic mice expressing IFN-γ in the epidermis develop inflammatory skin disease and features of SLE that have striking parallels with the human condition. We have now tested the effects of a pan-caspase inhibitor, carbobenzoxy-valyl-alanyl-aspartyl-(β-o-methyl)-fluoromethylketone, on disease progression. Daily s.c. administration of carbobenzoxy-valyl-alanyl-aspartyl-(β-o-methyl)-fluoromethylketone to female transgenic mice over a 3-wk period resulted in significant amelioration of both glomerular and interstitial renal damage, independent of the effects on autoantibody levels or skin inflammation. We propose that apoptosis inhibitors could be beneficial in the treatment of human SLE.
Technical Note: Linkage Disequilibrium and Disease-Associated CTLA4 Gene Polymorphisms
Authors: Holopainen Päivi M. , Partanen Jukka A.
Published: April 2014

CTLA4 and CD28 are important regulators of T lymphocyte activation. Gene region 2q33 carrying genes for both CTLA4 and CD28 has been shown to be linked to many autoimmune diseases. Disease associations with particular CTLA4 gene polymorphisms have been reported. Recently, first lines of evidence emerged for functional effects of CTLA4 gene polymorphisms. Two independent studies reported a reduced inhibitory function of CTLA4 in individuals with certain CTLA4 genotypes: those with a high number of microsatellite repeats in one study and those with allele +49*G in exon 1 in the other one. We analyzed the strength of linkage disequilibrium between the three known CTLA4 polymorphisms among 577 independent chromosomes. Our results show that the polymorphisms previously suggested to be the functional risk factors nearly always occur together in a very frequent haplotype. Due to this strong linkage disequilibrium, we conclude that the previous reports studying merely a single polymorphism could not distinguish which variation actually caused the functional difference. Hence, either mutagenesis approaches or studies with data on all linked polymorphisms are still needed to determine the genuine functional risk polymorphism in this gene region.
Role of CD47 in the Induction of Human Naive T Cell Anergy
Authors: Avice Marie-Noëlle , Rubio Manuel , Sergerie Martin , Delespesse Guy , Sarfati Marika
Published: April 2014

We recently reported that CD47 ligation inhibited IL-2 release by umbilical cord blood mononuclear cells activated in the presence of IL-12, but not IL-4, preventing the induction of IL-12Rβ2 expression and the acquisition of Th1, but not the Th2 phenotype. Here we show that in the absence of exogenous cytokine at priming, CD47 ligation of umbilical cord blood mononuclear cells promotes the development of hyporesponsive T cells. Naive cells were treated with CD47 mAb for 3 days, expanded in IL-2 for 9–12 days, and restimulated by CD3 and CD28 coengagement. Effector T cells generated under these conditions were considered to be anergic because they produced a reduced amount of IL-2 at the single-cell level and displayed an impaired capacity 1) to proliferate, 2) to secrete Th1/Th2 cytokines, and 3) to respond to IL-2, IL-4, or IL-12. Moreover, CD47 mAb strongly suppressed IL-2 production and IL-2Rα expression in primary cultures and IL-2 response of activated naive T cells. Induction of anergy by CD47 mAb was IL-10 independent, whereas inclusion of IL-2 and IL-4, but not IL-7, at priming fully restored T cell activation. Furthermore, CD28 costimulation prevented induction of anergy. Thus, CD47 may represent a potential target to induce anergy and prevent undesired Th0/Th1 responses such as graft vs host diseases, allograft rejection, or autoimmune diseases.
A CD19-Dependent Signaling Pathway Regulates Autoimmunity in Lyn-Deficient Mice
Authors: Hasegawa Minoru , Fujimoto Manabu , Poe Jonathan C. , Steeber Douglas A. , Lowell Clifford A. , Tedder Thomas F.
Published: April 2014

CD19 and the Src family protein tyrosine kinases (PTKs) are important regulators of intrinsic signaling thresholds in B cells. Regulation is achieved by cross-talk between Src family PTKs and CD19; Lyn is essential for CD19 phosphorylation, while CD19 establishes an Src family PTK activation loop that amplifies kinase activity. However, CD19-deficient (CD19−/−) B cells are hyporesponsive to transmembrane signals, while Lyn-deficient (Lyn−/−) B cells exhibit a hyper-responsive phenotype resulting in autoimmunity. To identify the outcome of interactions between CD19 and Src family PTKs in vivo, B cell function was examined in mice deficient for CD19 and Lyn (CD19/Lyn−/−). Remarkably, CD19 deficiency suppressed the hyper-responsive phenotype of Lyn−/− B cells and autoimmunity characterized by serum autoantibodies and immune complex-mediated glomerulonephritis in Lyn−/− mice. Consistent with Lyn and CD19 each regulating conventional B cell development, B1 cell development was markedly reduced by Lyn deficiency, with further reductions in the absence of CD19 expression. Tyrosine phosphorylation of Fyn and other cellular proteins induced following B cell Ag receptor ligation was dramatically reduced in CD19/Lyn−/− B cells relative to Lyn−/− B cells, while Syk phosphorylation was normal. In addition, the enhanced intracellular Ca2+ responses following B cell Ag receptor ligation that typify Lyn deficiency were delayed by the loss of CD19 expression. BCR-induced proliferation and humoral immune responses were also markedly inhibited by CD19/Lyn deficiency. These findings demonstrate that while the CD19/Lyn amplification loop is a major regulator of signal transduction thresholds in B lymphocytes, CD19 regulation of other Src family PTKs also influences B cell function and the development of autoimmunity.
The TNF Superfamily Members LIGHT and CD154 (CD40 Ligand) Costimulate Induction of Dendritic Cell Maturation and Elicit Specific CTL Activity
Authors: Morel Yannis , Truneh Alemseged , Sweet Raymond W. , Olive Daniel , Costello Régis T.
Published: April 2014

LIGHT is a recently identified member of the TNF superfamily that is up-regulated upon activation of T cells. Herpesvirus entry mediator, one of its receptors, is constitutively expressed on immature dendritic cells (DCs). In this report, we demonstrate that LIGHT induces partial DC maturation as demonstrated by Ag presentation and up-regulation of adhesion and costimulatory molecules. LIGHT-stimulated DCs show reduced macropinocytosis and enhanced allogeneic stimulatory capacity but fail to produce significant amounts of IL-12, IL-6, IL-1β, or TNF-α compared with unstimulated DCs. However, LIGHT cooperates with CD154 (CD40 ligand) in DC maturation, with particular potentiation of allogeneic T cell proliferation and cytokine secretion of IL-12, IL-6, and TNF-α. Moreover, LIGHT costimulation allows DCs to prime in vitro-enhanced specific CTL responses. Our results suggest that LIGHT plays an important role in DC-mediated immune responses by regulating CD154 signals and represents a potential tool for DC-based cancer immunotherapy.
IFN-γ Induces the Apoptosis of WEHI 279 and Normal Pre-B Cell Lines by Expressing Direct Inhibitor of Apoptosis Protein Binding Protein with Low pI
Authors: Yoshikawa Hideshi , Nakajima Yasuo , Tasaka Kachio
Published: April 2014

Interferon-γ plays a crucial role in induction of Th1 response but is predominantly a negative regulator of B cell differentiation and Th2 response, so it is a key molecule in determining cellular or humoral immunity. In this study, we demonstrate that IFN-γ induces apoptosis in WEHI 279 mouse B cells and IL-7-dependent mouse pre-B cells by disrupting mitochondrial membrane potential and cytochrome c release via down-regulation of Bcl-2 and Bcl-xL. Furthermore, this apoptotic signal is promoted by the de novo synthesis of endogenous direct inhibitor of apoptosis protein binding protein with low pI (DIABLO) by IFN-γ and its release from mitochondria into the cytosol. Inhibition of DIABLO expression by antisense oligonucleotide is sufficient to decrease caspase activities and DNA fragmentation, but not cytochrome c release from mitochondria, suggesting that DIABLO plays a critical role in promoting apoptotic signals downstream of mitochondrial events. Thus, these findings demonstrate a signaling pathway during B cell apoptosis induced by IFN-γ and possible mechanisms by which B cell differentiation is negatively regulated by Th1-type cytokines.
Double-Stranded RNA Regulates IL-4 Expression
Authors: Kehoe Kelly E. , Brown Melissa A. , Imani Farhad
Published: April 2014

dsRNA, as genomic fragment, replicative intermediate, or stem and loop structure in cells infected by viruses, can act to signal the immune system of the presence of viral infections. Although most viral infections are associated with strong Th1 immune responses, Th2-type responses have also been observed. In this study, we characterize the effects of dsRNA on the induction of Th2 responses in human lymphocytes. We report that in addition to the well-known Th1-inducing capabilities of dsRNA, treatment of human lymphocytes with low concentrations of dsRNA (0.1–1 μg/ml) leads to the expression of the prototypic Th2 cytokine IL-4. This induction was accompanied with the concentration-dependent activation of NF-κB and NF-AT2 but not NF-AT1. In addition, dsRNA can directly activate an IL-4 promoter-driven chloramphenicol acetyltransferase reporter gene in transiently transfected Jurkat cells. These results are the first demonstration of a non-TCR-associated activator of NF-AT in human cells and suggest that dsRNA directly influences IL-4 gene expression through its effect on NF-AT activation. Our data provide support for the idea that dsRNA at low concentrations in vivo may induce a Th2-dominant response that is not optimal for protective immunity to the virus.
Suppression of Granulocyte-Macrophage Colony-Stimulating Factor Expression by Glucocorticoids Involves Inhibition of Enhancer Function by the Glucocorticoid Receptor Binding to Composite NF-AT/Activator Protein-1 Elements
Authors: Smith Philip J. , Cousins David J. , Jee Young-Koo , Staynov Dontcho Z. , Lee Tak H. , Lavender Paul
Published: April 2014

Increased expression of a number of cytokines including GM-CSF is associated with chronic inflammatory conditions such as bronchial asthma. Glucocorticoid therapy results in suppression of cytokine levels by a mechanism(s) not yet fully understood. We have examined regulation of GM-CSF expression by the synthetic glucocorticoid dexamethasone in human T cells. Transient transfection assays with reporter constructs revealed that dexamethasone inhibited the function of the GM-CSF enhancer, but had no effect on regulation of GM-CSF expression occurring through the proximal promoter. Activation of the GM-CSF enhancer involves cooperative interaction between the transcription factors NF-AT and AP-1. We demonstrate here that glucocorticoid-mediated inhibition of enhancer function involves glucocorticoid receptor (GR) binding to the NF-AT/AP-1 sites. These elements, which do not constitute recognizable glucocorticoid response elements, support binding of the GR, primarily as a dimer. This binding correlates with the ability of dexamethasone to inhibit enhancer activity of the NF-AT/AP-1 elements, suggesting a competition between NF-AT/AP-1 proteins and GR.
Expression of α4β7 Integrin Defines a Distinct Pathway of Lymphoid Progenitors Committed to T Cells, Fetal Intestinal Lymphotoxin Producer, NK, and Dendritic Cells
Authors: Yoshida Hisahiro , Kawamoto Hiroshi , Santee Sybil M. , Hashi Hiroyuki , Honda Kenya , Nishikawa Satomi , Ware Carl F. , Katsura Yoshimoto , Nishikawa Shin-Ichi
Published: April 2014

During embryogenesis, the Peyer’s patch anlagen are induced by a cell population that produces lymphotoxin (LT) α1β2 following stimulation of IL-7Rα. In this study, we show that the LT-producing cell is localized within the IL-7Rα+ and integrin α4β7 (α4β7)+ population in the embryonic intestine. Lineage commitment to the LT producer phenotype in the fetal liver coincides with expression of α4β7. Before expression of α4β7, the potential of IL-7Rα+ population to generate B cells is lost. However, the progenitors for T cells and LT producer cells reside in the IL-7Rα+α4β7+ cells, but during subsequent differentiation, the potential to give rise to T cells is lost. This IL-7Rα+α4β7+ population migrates to the intestine, where it induces the Peyer’s patch anlagen. When stimulated with IL-15 or IL-3 and TNF, the intestinal IL-7Rα+α4β7+ population can differentiate into fully competent NK1.1+ NK cells or CD11c+ APCs. Expression of α4β7 is lost during differentiation of both lineages; IL-7Rα expression is lost during NK1.1+ cells differentiation. A newly discovered lineage−IL-7Rα+c-Kit+α4β7+ population in the fetal liver is committed to T, NK, dendritic, and fetal intestinal LT producer lineage, the latter being an intermediate stage during differentiation of NK and dendritic cells.
Longevity of Antigen Presentation and Activation Status of APC Are Decisive Factors in the Balance Between CTL Immunity Versus Tolerance
Authors: Th. den Boer Annemieke , Diehl Linda , van Mierlo Geertje J. D. , van der Voort Ellen I. H. , Fransen Marieke F. , Krimpenfort Paul , Melief Cornelis J. M. , Offringa Rienk , Toes René E. M.
Published: April 2014

Encounter of Ag by naive T cells can lead to T cell priming as well as tolerance. The balance between immunity and tolerance is controlled by the conditions of Ag encounter and the activation status of the APC. We have investigated the rules that govern this balance in case an environment that normally induces tolerance is reverted into a milieu that promotes T cell priming, using a minimal CTL epitope derived from human adenovirus type 5 E1A. Vaccination of mice s.c. with E1A peptide in IFA readily induces CTL tolerance, resulting in the inability to control E1A-expressing tumors. The present study shows that efficient CTL priming is achieved when this peptide vaccine is combined with systemic administration of APC-activating compounds like agonistic anti-CD40 mAb or polyriboinosinate-polyribocytidylate. Surprisingly, this CTL response is not long-lasting and therefore fails to protect against tumor outgrowth. Disappearance of CTL reactivity was strongly associated with systemic persistence of the peptide for >200 days. In contrast, peptide administered in PBS does not persist and generates long term CTL immunity capable of rejecting Ad5E1A-positive tumors, when combined with CD40 triggering. Thus, presentation of CTL epitopes in an appropriate costimulatory setting by activated APC, although being essential and sufficient for CTL priming, eventually results in tolerance when the Ag persists systemically for prolonged times. These observations are important for the development of immune intervention schemes in autoimmunity and cancer.
Exogenous Peptides Presented by Transporter Associated with Antigen Processing (TAP)-Deficient and TAP-Competent Cells: Intracellular Loading and Kinetics of Presentation
Authors: Luft Thomas , Rizkalla Mark , Tai Tsin Yee , Chen Qiyuan , MacFarlan Roderick I. , Davis Ian D. , Maraskovsky Eugene , Cebon Jonathan
Published: April 2014

This study investigates the differential capacity of TAP-deficient T2 cells, TAP-competent EBV cells, and immature and mature dendritic cells to present peptides to preformed CTL lines. It demonstrates that presentation of exogenous peptides involves peptide uptake and loading onto newly synthesized MHC class I molecules. This mechanism was best demonstrated for low affinity peptides in the presence of irrelevant peptides competing for HLA binding sites. Under these circumstances, inhibition of protein synthesis with cycloheximide or vesicular trafficking with brefeldin A significantly reduced the presentation of low affinity peptides. This was not restored by adding exogenous β2-microglobulin to stabilize the MHC complex on the cell surface. In contrast, presentation of high affinity peptides was not sensitive to cycloheximide or brefeldin A, which suggests that different mechanisms may operate for presentation of high and low affinity peptides by TAP-competent cells. High affinity peptides can apparently compete with peptides in preloaded MHC class I molecules at the cell surface, whereas low affinity peptides require empty MHC molecules within cells. Accordingly, very high concentrations of exogenous low affinity peptides in conjunction with active MHC class I metabolism were required to allow successful presentation against a background of competing intracellular high affinity peptides in TAP-competent cells. These findings have implications for the design of peptide and protein-based vaccines.
Neutrophils Process Exogenous Bacteria Via an Alternate Class I MHC Processing Pathway for Presentation of Peptides to T Lymphocytes
Authors: Potter Nicholas S. , Harding Clifford V.
Published: April 2014

Peptides that are presented by class I MHC (MHC-I) molecules derive from cytosolic Ags processed via the conventional MHC-I pathway or exogenous Ags processed via alternate MHC-I processing mechanisms. Alternate MHC-I processing by macrophages and dendritic cells allows presentation of peptides from particulate Ags, including bacteria. Despite the established phagocytic activity of neutrophils, MHC-I processing and presentation of phagocytosed Ags by neutrophils has not been investigated. Murine neutrophils from peritoneal exudates were shown to express MHC-I molecules and tested for the ability to process HB101.Crl-OVA, Escherichia coli transfected to express a fusion protein containing the 257–264 epitope of OVA. Neutrophils were found to process HB101.Crl-OVA and present OVA257–264-Kb complexes to CD8OVA T hybridoma cells via a pathway that was resistant to brefeldin A, an inhibitor of anterograde endoplasmic reticulum-Golgi transport, and lactacystin, a proteasome inhibitor. These results suggest that neutrophils process phagocytosed bacteria via a vacuolar alternate MHC-I pathway that does not involve cytosolic processing. In addition, neutrophils were found to secrete or “regurgitate” processed peptide that was subsequently presented by neighboring prefixed macrophages or dendritic cells. Thus, neutrophils may influence T cell responses to bacteria, either by directly presenting peptide-MHC-I complexes or by delivering peptides to other APCs for presentation. Hypothetically, neutrophils may directly present peptide to effector T cells in vivo at sites of inflammation, inducing cytokine production, whereas dendritic cells in receipt of neutrophil-derived antigenic peptides may migrate to lymphoid organs to initiate T cell responses.
Bidirectional Negative Regulation of Human T and Dendritic Cells by CD47 and Its Cognate Receptor Signal-Regulator Protein-α: Down-Regulation of IL-12 Responsiveness and Inhibition of Dendritic Cell Activation
Authors: Latour Sylvain , Tanaka Hiroyuki , Demeure Christian , Mateo Véronique , Rubio Manuel , Brown Eric J. , Maliszewski Charles , Lindberg Frederik P. , Oldenborg Anna , Ullrich Axel , Delespesse Guy , Sarfati Marika
Published: April 2014

Proinflammatory molecules, including IFN-γ and IL-12, play a crucial role in the elimination of causative agents. To allow healing, potent anti-inflammatory processes are required to down-regulate the inflammatory response. In this study, we first show that CD47/integrin-associated protein, a ubiquitous multispan transmembrane protein highly expressed on T cells, interacts with signal-regulator protein (SIRP)-α, an immunoreceptor tyrosine-based inhibition motif-containing molecule selectively expressed on myelomonocytic cells, and next demonstrate that this pair of molecules negatively regulates human T and dendritic cell (DC) function. CD47 ligation by CD47 mAb or L-SIRP-α transfectants inhibits IL-12R expression and down-regulates IL-12 responsiveness of activated CD4+ and CD8+ adult T cells without affecting their response to IL-2. Human CD47-Fc fusion protein binds SIRP-α expressed on immature DC and mature DC. SIRP-α engagement by CD47-Fc prevents the phenotypic and functional maturation of immature DC and still inhibits cytokine production by mature DC. Finally, in allogeneic MLR between mDC and naive T cells, CD47-Fc decreases IFN-γ production after priming and impairs the development of a Th1 response. Therefore, CD47 on T cells and its cognate receptor SIRP-α on DC define a novel regulatory pathway that may be involved in the maintenance of homeostasis by preventing the escalation of the inflammatory immune response.
Antigen-Specific Blockade of T Cells In Vivo Using Dimeric MHC Peptide
Authors: O’Herrin Sean M. , Slansky Jill E. , Tang Q. , Markiewicz Mary A. , Gajewski Thomas F. , Pardoll Drew M. , Schneck Jonathan P. , Bluestone Jeffrey A.
Published: April 2014

Ag-specific immune tolerance in clinical organ transplantation is currently an unrealized but critical goal of transplant biology. The specificity and avidity of multimerized MHC-peptide complexes suggests their potential ability to modulate T cell sensitization and effector functions. In this study, we examined the ability of MHC-peptide dimers to modulate T cell function both in vitro and in vivo. Soluble MHC dimers induced modulation of surface TCR expression and inhibited T cell cytolytic activity at nanomolar concentrations in vitro. Furthermore, engagement of TCR by soluble dimers resulted in phosphorylation of the TCR ζ-chain and recruitment and phosphorylation of ζ-associated protein-70 to the signaling complex, the latter of which increased upon dimer cross-linking. Significantly, Ag-specific inhibition of an alloreactive TCR-transgenic T cell population in vivo resulted in consequent outgrowth of an allogeneic tumor. The prolonged Ag-specific suppression of expansion and/or effector function of cognate T cells in vivo suggests that soluble MHC dimers may be a means of inducing sustained Ag-specific T cell unresponsiveness in vivo.
MHC Class I Allele Dosage Alters CD8 Expression by Intestinal Intraepithelial Lymphocytes
Authors: Podd Bradley S. , Åberg Caroline , Kudla Kimberly L. , Keene Lataya , Tobias Erin , Camerini Victoria
Published: April 2014

The development of TCR αβ+, CD8αβ+ intestinal intraepithelial lymphocytes (IEL) is dependent on MHC class I molecules expressed in the thymus, while some CD8αα+ IEL may arise independently of MHC class I. We examined the influence of MHC I allele dosage on the development CD8+ T cells in RAG 2−/− mice expressing the H-2Db-restricted transgenic TCR specific for the male, Smcy-derived H-Y Ag (H-Y TCR). IEL in male mice heterozygous for the restricting (H-2Db) and nonrestricting (H-2Dd) MHC class I alleles (MHC F1) were composed of a mixture of CD8αβ+ and CD8αα+ T cells, while T cells in the spleen were mostly CD8αβ+. This was unlike IEL in male mice homozygous for H-2Db, which had predominantly CD8αα+ IEL and few mostly CD8− T cells in the spleen. Our results demonstrate that deletion of CD8αβ+ cells in H-Y TCR male mice is dependent on two copies of H-2Db, whereas the generation of CD8αα+ IEL requires only one copy. The existence of CD8αβ+ and CD8αα+ IEL in MHC F1 mice suggests that their generation is not mutually exclusive in cells with identical TCR. Furthermore, our data imply that the level of the restricting MHC class I allele determines a threshold for conventional CD8αβ+ T cell selection in the thymus of H-Y TCR-transgenic mice, whereas the development of CD8αα+ IEL is dependent on, but less sensitive to, this MHC class I allele.
Immune Rejection of a Large Sarcoma Following Cyclophosphamide and IL-12 Treatment Requires Both NK and NK T Cells and Is Associated with the Induction of a Novel NK T Cell Population
Authors: Karnbach Claudia , Daws Michael R. , Niemi Eréne C. , Nakamura Mary C.
Published: April 2014

Combined immunotherapy with cyclophosphamide (Cy) and IL-12, but not IL-12 alone, stimulates eradication of a large established solid tumor (20 mm), MCA207, a methylcholanthrene-induced murine sarcoma. In these studies we demonstrate that NK1.1+ cells and CD1d-dependent NK T cells each play important yet distinct roles in regression of a large tumor in response to Cy and IL-12, and we define a novel NK T cell subset, selectively increased by this treatment. Mice depleted of NK1.1+ cells demonstrated more rapid initial tumor growth and prolonged tumor regression following treatment, but tumors were eventually eradicated. In contrast, initial tumor regression following therapy was unimpaired in CD1d−/− mice, which are deficient in most NK T cells, but tumors recurred. No tumor regression occurred following Cy and IL-12 therapy in CD1d−/− mice that were depleted of NK1.1+ cells. We found that Cy and IL-12 induced the selective increase in liver and spleen lymphocytes of a unique NK T subpopulation (DX5+NK1.1−CD3+). These cells were not induced by treatment in CD1d−/− mice. Our studies demonstrate a contribution of both NK and NK T cells to the Cy- and IL-12-stimulated anti-tumor response. We describe the selective induction of a distinct NK T cell subset by Cy and IL-12 therapy, not seen following IL-12 therapy alone, which we suggest may contribute to the successful anti-tumor response induced by this immunotherapeutic regimen.
Peptide Requirement for CTL Activation Reflects the Sensitivity to CD3 Engagement: Correlation with CD8αβ Versus CD8αα Expression
Authors: Cawthon Andrew G. , Lu Haiping , Alexander-Miller Martha A.
Published: April 2014

In our previous studies, CTL that were sensitive to low concentrations of peptide Ag were found to be far superior to those requiring high concentrations of Ag for reducing viral burden when adoptively transferred into SCID mice. Thus it is important that we understand the mechanisms that control the requirement for peptide Ag with the long-term goal of selectively expanding these exquisitely sensitive cells in vivo. Although TCR affinity is one parameter that can affect the CTL sensitivity for Ag, we investigated whether additional mechanisms may also be involved. In studies using a TCR transgenic mouse model, we successfully generated CTL with identical TCR affinity that possess distinctly different activation requirements. Using both peptide Ag and anti-CD3 Ab to activate the CTL lines of high vs low avidity, we found that the variations in activation threshold are the result of differences in the required number of engaged TCR. Additionally, we have observed that the ratio of CD8αβ to CD8αα is significantly greater in CTL lines that are more sensitive to TCR engagement, which may contribute to the lower activation threshold of these CTL following CD3 engagement. These studies identify a novel mechanism by which the activation requirements of Ag-specific CTL are determined by demonstrating a direct correlation between the sensitivity to TCR engagement, the expression of levels CD8αβ vs αα, and the amount of peptide Ag required to reach the threshold for activation.