bluuebunny/crossref_metadata_2025 huggingface dataset:
Signaling Lymphocytic Activation Molecule Expression and Regulation in Human Intracellular Infection Correlate with Th1 Cytokine Patterns
Authors: García Verónica E. , Quiroga María F. , Ochoa María T. , Ochoa Laura , Pasquinelli Virginia , Fainboim Leonardo , Olivares Liliana M. , Valdez Raúl , Sordelli Daniel O. , Aversa Gregorio , Modlin Robert L. , Sieling Peter A.
Published: April 2014

Induction of Th1 cytokines, those associated with cell-mediated immunity, is critical for host defense against infection by intracellular pathogens, including mycobacteria. Signaling lymphocytic activation molecule (SLAM, CD150) is a transmembrane protein expressed on lymphocytes that promotes T cell proliferation and IFN-γ production. The expression and role of SLAM in human infectious disease were investigated using leprosy as a model. We found that SLAM mRNA and protein were more strongly expressed in skin lesions of tuberculoid patients, those with measurable CMI to the pathogen, Mycobacterium leprae, compared with lepromatous patients, who have weak CMI against M. leprae. Peripheral blood T cells from tuberculoid patients showed a striking increase in the level of SLAM expression after stimulation with M. leprae, whereas the expression of SLAM on T cells from lepromatous patients show little change by M. leprae stimulation. Engagement of SLAM by an agonistic mAb up-regulated IFN-γ production from tuberculoid patients and slightly increased the levels of IFN-γ in lepromatous patients. In addition, IFN-γ augmented SLAM expression on M. leprae-stimulated peripheral blood T cells from leprosy patients. Signaling through SLAM after IFN-γ treatment of Ag-stimulated cells enhanced IFN-γ production in lepromatous patients to the levels of tuberculoid patients. Our data suggest that the local release of IFN-γ by M. leprae-activated T cells in tuberculoid leprosy lesions leads to up-regulation of SLAM expression. Ligation of SLAM augments IFN-γ production in the local microenvironment, creating a positive feedback loop. Failure of T cells from lepromatous leprosy patients to produce IFN-γ in response to M. leprae contributes to reduced expression of SLAM. Therefore, the activation of SLAM may promote the cell-mediated immune response to intracellular bacterial pathogens.
Graphical Representation of a Generalized Linear Model-Based Statistical Test Estimating the Fit of the Single-Hit Poisson Model to Limiting Dilution Assays
Authors: Bonnefoix Thierry , Bonnefoix Philippe , Callanan Mary , Verdiel Paul , Sotto Jean-Jacques
Published: April 2014

Standardized statistical and graphical methods for analysis of limiting dilution assays are highly desirable to enable investigators to compare and interpret results and conclusions with greater accuracy and precision. According to these requirements, we present in this work a powerful statistical slope test that estimates the fit of the single-hit Poisson model to limiting dilution experiments. This method is readily amenable to a graphical representation. This slope test is obtained by modeling limiting dilution data according to a linear log-log regression model, which is a generalized linear model specially designed for modeling binary data. The result of the statistical slope test can then be graphed to visualize whether the data are compatible or not with the single-hit Poisson model. We demonstrate this statistical test and its graphical representation by using two examples: a real limiting dilution experiment evaluating the growth frequency of IL-2-responsive tumor-infiltrating T cells in a malignant lymph node involved by a B cell non-Hodgkin’s lymphoma, and a simulation of a limiting dilution assay corresponding to a theoretical non-single-hit Poisson model, suppressor two-target Poisson model.
Epitope Mapping Using the X-Ray Crystallographic Structure of Complement Receptor Type 2 (CR2)/CD21: Identification of a Highly Inhibitory Monoclonal Antibody That Directly Recognizes the CR2-C3d Interface
Authors: Guthridge Joel M. , Young Kendra , Gipson Matthew G. , Sarrias Maria-Rossa , Szakonyi Gerda , Chen Xiaojiang S. , Malaspina Angela , Donoghue Eileen , James Judith A. , Lambris John D. , Moir Susan A. , Perkins Stephen J. , Holers V. Michael
Published: April 2014

Complement receptor type 2 (CR2)/CD21 is a B lymphocyte cell membrane C3d/iC3b receptor that plays a central role in the immune response. Human CR2 is also the receptor for the EBV viral membrane glycoprotein gp350/220. Both C3d and gp350/220 bind CR2 within the first two of 15–16 repetitive domains that have been designated short consensus/complement repeats. Many mAbs react with human CR2; however, only one currently available mAb is known to block both C3d/iC3b and gp350/220 binding. We have used a recombinant form of human CR2 containing the short consensus/complement repeat 1-2 ligand-binding fragment to immunize Cr2−/− mice. Following fusion, we identified and further characterized four new anti-CR2 mAbs that recognize this fragment. Three of these inhibited binding of CR2 to C3d and gp350/220 in different forms. We have determined the relative inhibitory ability of the four mAbs to block ligand binding, and we have used overlapping peptide-based approaches to identify linear epitopes recognized by the inhibitory mAbs. Placement of these epitopes on the recently solved crystal structure of the CR2-C3d complex reveals that each inhibitory mAb recognizes a site either within or adjacent to the CR2-C3d contact site. One new mAb, designated 171, blocks CR2 receptor-ligand interactions with the greatest efficiency and recognizes a portion of the C3d contact site on CR2. Thus, we have created an anti-human CR2 mAb that blocks the C3d ligand by direct contact with its interaction site, and we have provided confirmatory evidence that the C3d binding site seen in its crystal structure exists in solution.
Association of FcγRII with Low-Density Detergent-Resistant Membranes Is Important for Cross-Linking-Dependent Initiation of the Tyrosine Phosphorylation Pathway and Superoxide Generation
Authors: Katsumata Osamu , Hara-Yokoyama Miki , Sautès-Fridman Catherine , Nagatsuka Yasuko , Katada Toshiaki , Hirabayashi Yoshio , Shimizu Kazufumi , Fujita-Yoshigaki Junko , Sugiya Hiroshi , Furuyama Shunsuke
Published: April 2014

IgG immune complexes trigger humoral immune responses by cross-linking of FcRs for IgG (FcγRs). In the present study, we investigated role of lipid rafts, glycolipid- and cholesterol-rich membrane microdomains, in the FcγR-mediated responses. In retinoic acid-differentiated HL-60 cells, cross-linking of FcγRs resulted in a marked increase in the tyrosine phosphorylation of FcγRIIa, p58lyn, and p120c-cbl, which was inhibited by a specific inhibitor of Src family protein tyrosine kinases. After cross-linking, FcγRs and tyrosine-phosphorylated proteins including p120c-cbl were found in the low-density detergent-resistant membrane (DRM) fractions isolated by sucrose-density gradient ultracentrifugation. The association of FcγRs as well as p120c-cbl with DRMs did not depend on the tyrosine phosphorylation. When endogenous cholesterol was reduced with methyl-β-cyclodextrin, the cross-linking did not induce the association of FcγRs as well as p120c-cbl with DRMs. In addition, although the physical association between FcγRIIa and p58lyn was not impaired, the cross-linking did not induce the tyrosine phosphorylation. In human neutrophils, superoxide generation induced by opsonized zymosan or chemoattractant fMLP was not affected or increased, respectively, after the methyl-β-cyclodextrin treatment, but the superoxide generation induced by the insoluble immune complex via FcγRII was markedly reduced. Accordingly, we conclude that the cross-linking-dependent association of FcγRII to lipid rafts is important for the activation of FcγRII-associated Src family protein tyrosine kinases to initiate the tyrosine phosphorylation cascade leading to superoxide generation.
A Common Single Nucleotide Polymorphism in the CD14 Promoter Decreases the Affinity of Sp Protein Binding and Enhances Transcriptional Activity
Authors: LeVan Tricia D. , Bloom John W. , Bailey Thomas J. , Karp Christopher L. , Halonen Marilyn , Martinez Fernando D. , Vercelli Donata
Published: April 2014

CD14 is a pattern recognition receptor that plays a central role in innate immunity through recognition of bacterial lipoglycans, primarily LPS. Recently, our group has identified a common single nucleotide polymorphism, −159C→T, in the CD14 proximal promoter. Homozygous carriers of the T allele have a significant increase in soluble CD14, but a decreased total serum IgE. This epidemiologic evidence led us to investigate the molecular basis for the effects of CD14/−159C→T on CD14 regulation in monocytes and hepatocytes, the two major cell types known to express this gene in vivo. EMSA analysis showed that the T allele results in decreased affinity of DNA/protein interactions at a GC box that contains a binding site for Sp1, Sp2, and Sp3 transcription factors. In reporter assays, the transcriptional activity of the T allele was increased in monocytic Mono Mac 6 cells, which express low levels of Sp3, a member of the Sp family with inhibitory potential relative to activating Sp1 and Sp2. By contrast, both alleles were transcribed equivalently in Sp3-rich hepatocytic HepG2 cells. Our data indicate that the interplay between CD14 promoter affinity and the [Sp3]:[Sp1 + Sp2] ratio plays a critical mechanistic role in regulating transcription of the two CD14 alleles. Variation in a key gene of innate immunity may be important for the pathogenesis of allergy and inflammatory disease through gene-by-gene and/or gene-by-environment interactions.
Inability of IL-12 to Down-Regulate IgE Synthesis Due to Defective Production of IFN-γ in Atopic NC/Nga Mice
Authors: Matsumoto Masahiro , Itakura Atsuko , Tanaka Akane , Fujisawa Chie , Matsuda Hiroshi
Published: April 2014

NC/Nga mice raised in nonsterile circumstances spontaneously suffer from atopic dermatitis-like skin lesions with IgE hyperproduction. We investigated effects of rIL-12 on the IgE production in NC/Nga mice. rIL-12 administration was successful to suppress the increase of IgE levels in BALB/c mice immunized with OVA and aluminum hydroxide, but failed to abrogate that in NC/Nga mice. Both in vivo and in vitro IFN-γ production induced by rIL-12 was less in NC/Nga mice than in BALB/c mice. Addition of rIFN-γ to rIL-4 and LPS completely abrogated IgE production by B cells of BALB/c mice, but was insufficient to suppress it by B cells of NC/Nga mice. In splenic cells pretreated with Con A, STAT4 was phosphorylated at the tyrosine residue by addition of rIL-12, which was more weakly inducible in NC/Nga mice than in BALB/c mice. Finally, we examined the preventive ability of rIL-12 on the clinical aspects of atopic dermatitis in NC/Nga mice. rIL-12 administration resulted in exacerbation of development of the skin lesions and IgE production in NC/Nga mice raised in nonsterile circumstances. These results suggest that defective production of IFN-γ by T cells less sensitive to IL-12 and low responsiveness of B cells to IFN-γ may contribute to IgE hyperproduction in NC/Nga mice, and that IL-12 may have no ability to improve the clinical aspects of NC/Nga mice.
Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-κB, c-Myc, and pRb Pathways
Authors: Buggins Andrea G. S. , Milojkovic Dragana , Arno Matthew J. , Lea Nicholas C. , Mufti Ghulam J. , Thomas N. Shaun B. , Hirst William J. R.
Published: April 2014

Tumors produce a variety of immunosuppressive factors which can prevent the proliferation and maturation of a number of normal hemopoietic cell types. We have investigated whether primary acute myeloid leukemia (AML) cells have an effect on normal T cell function and signaling. Tumor cell supernatant (TSN) from AML cells inhibited T cell activation and Th1 cytokine production and also prevented activated T cells from entering the cell cycle. These effects occurred in the absence of AML cell-T cell contact. We have demonstrated that AML TSN contained none of the immunosuppressors described to date, namely gangliosides, nitric oxide, TGF-β, IL-10, vascular endothelial growth factor, or PGs. Furthermore, IL-2 did not overcome the block, despite normal IL-2R expression. However, the effect was overcome by preincubation with inhibitors of protein secretion and abolished by trypsinization, indicating that the active substance includes one or more proteins. To determine the mechanism of inhibition, we have studied many of the major pathways involved in T cell activation and proliferation. We show that nuclear translocation of NFATc and NF-κB are markedly reduced in T cells activated in the presence of primary AML cells. In contrast, calcium mobilization and activation of other signal transduction pathways, namely extracellular signal-regulated kinase1/2, p38, and STAT5 were unaffected, but activation of c-Jun N-terminal kinase 1/2 was delayed. Phosphorylation of pRb by cyclin-dependent kinase 6/4-cyclin D and of p130 did not occur and c-Myc, cyclin D3, and p107 were not induced, consistent with cell cycle inhibition early during the transition from G0 to G1. Our data indicate that TSN generated by AML cells induces T cell immunosuppression and provides a mechanism by which the leukemic clone could evade T cell-mediated killing.
Pre-Emptive Targeting of the Epitope Spreading Cascade with Genetically Modified Regulatory T Cells During Autoimmune Demyelinating Disease
Authors: Yin Ling , Yu Min , Edling Andrea E. , Kawczak Julie A. , Mathisen Peter M. , Nanavati Tania , Johnson Justin M. , Tuohy Vincent K.
Published: April 2014

Epitope spreading or endogenous self-priming has been implicated in mediating the progression of autoimmune disease. In the present study we created an immune-deviated, epitope spreading response in SWXJ mice after the onset of experimental autoimmune encephalomyelitis, a prototypic autoimmune animal model widely used in multiple sclerosis research. We established an immunoregulatory spreading repertoire by transferring T cells genetically modified to produce high levels of IL-10 in response to a dominant epitope spreading determinant. Installation of a Th2/Tr1-like spreading repertoire resulted in a marked and prolonged inhibition of disease progression and demyelination characterized by 1) bystander inhibition of the recall response to the priming immunogen, and 2) a Th1→Tr1 immune-deviated spreading response involving a shift in the source of IL-10 production from the transferred regulatory population to the host-derived, endogenously primed repertoire. Thus, our data provide a rationale for cell-based therapeutic intervention in multiple sclerosis by showing that pre-emptive targeting of the epitope spreading cascade with regulatory T cells effectively induces an immune-deviated spreading response capable of inhibiting ongoing inflammatory autoreactivity and disease progression.
Differential Effects of Physiologically Relevant Hypoxic Conditions on T Lymphocyte Development and Effector Functions
Authors: Caldwell Charles C. , Kojima Hidefumi , Lukashev Dmitriy , Armstrong John , Farber Mark , Apasov Sergey G. , Sitkovsky Michail V.
Published: April 2014

Direct measurements revealed low oxygen tensions (0.5–4.5% oxygen) in murine lymphoid organs in vivo. To test whether adaptation to changes in oxygen tension may have an effect on lymphocyte functions, T cell differentiation and functions at varying oxygen tensions were studied. These studies show: 1) differentiated CTL deliver Fas ligand- and perforin-dependent lethal hit equally well at all redox conditions; 2) CTL development is delayed at 2.5% oxygen as compared with 20% oxygen. Remarkably, development of CTL at 2.5% oxygen is more sustained and the CTL much more lytic; and 3) hypoxic exposure and TCR-mediated activation are additive in enhancing levels of hypoxia response element-containing gene products in lymphocyte supernatants. In contrast, hypoxia inhibited the accumulation of nonhypoxia response element-containing gene products (e.g., IL-2 and IFN-γ) in the same cultures. This suggests that T cell activation in hypoxic conditions in vivo may lead to different patterns of lymphokine secretion and accumulation of cytokines (e.g., vascular endothelial growth factor) affecting endothelial cells and vascular permeabilization. Thus, although higher numbers of cells survive and are activated during 20% oxygen incubation in vitro, the CTL which develop at 2.5% oxygen are more lytic with higher levels of activation markers. It is concluded that the ambient 20% oxygen tension (plus 2-ME) is remarkably well suited for immunologic specificity and cytotoxicity studies, but oxygen dependence should be taken into account during the design and interpretation of results of in vitro T cell development assays and gene expression studies in vivo.
CD8+ T Lymphocytes in Double αβ TCR Transgenic Mice. II. Competitive Fitness of Dual αβ TCR CD8+ T Lymphocytes in the Peripheral Pools
Authors: Legrand Nicolas , Freitas Antonio A.
Published: April 2014

We studied Rag2-deficient mice bearing two rearranged αβ TCR transgenes, both restricted to the MHC H-2Db class I molecule. We have previously shown that, in these DTg mice, most peripheral CD8 T cells express one TCRβ chain associated with two TCRα chains, as in one-third of the mature T cells from normal mice. We examined the functional behavior of the dual-receptor CD8 T cells developing either in the absence or in the presence of self-Ag. The dual-receptor CD8 T cells, which develop in absence of self-Ag, show efficient responses to immunization and remain sensitive to induction of peripheral tolerance. In contrast to single TCR T cells, the dual-TCR cells, when tolerized upon exposure to high levels of self-Ag, are not deleted and therefore may exert important regulatory functions. When developing in the presence of self-Ag, the dual-receptor-expressing CD8 T cells escape central deletion, but are not fully competent to respond to cognate stimuli. Overall, we found that the dual-TCR CD8 T cells show a poor competitive value and can be out-competed by single-TCR cells, both in the course of immune responses and in reconstitution experiments. The decreased fitness of the dual-receptor cells may contribute to diminishing the autoimmune hazard that they could represent.
TCR/Self-Antigen Interactions Drive Double-Negative T Cell Peripheral Expansion and Differentiation into Suppressor Cells
Authors: Priatel John J. , Utting Oliver , Teh Hung-Sia
Published: April 2014

Mature CD4−CD8− αβ+ T cells (DNTC) in the periphery of TCR transgenic mice are resistant to clonal deletion in cognate Ag-expressing (Ag+) mice. Previously, we have characterized DNTC populations bearing the alloreactive 2C TCR in Ag-free (Ag−) and Ag+ mice. Despite appearing functionally anergic when challenged with cognate Ag in vitro, Ag-experienced DNTC exhibit markers of activation/memory, a lowered threshold of activation, ex vivo cytolytic activity, and the ability to rapidly secrete IFN-γ. Remarkably, these memory-like DNTC also possess potent immunoregulatory properties, competing effectively for bystander-produced IL-2 and suppressing autoreactive CD8+ T cell proliferation via a Fas/FasL-dependent cytolytic mechanism. The fact that DNTC recovered from Ag+ mice possess markers and attributes characteristic of naive CD8+ T cells that have undergone homeostasis-induced proliferation suggested that they may be derived from a similar peripheral expansion process. Naive DNTC adoptively transferred into Ag-bearing hosts rapidly acquire markers and functional attributes of DNTC that have continually developed in the presence of Ag. Thus, the peripheral selection and maintenance of such autoreactive cells may serve to negatively regulate potential autoimmune T cell responses.
The IL-15Rα Chain Signals Through Association with Syk in Human B Cells
Authors: Bulanova Elena , Budagian Vadim , Pohl Thomas , Krause Hans , Dürkop Horst , Paus Ralf , Bulfone-Paus Silvia
Published: April 2014

The α-chain of the IL-15R (IL-15Rα) serves as the specific, high-affinity receptor for IL-15. It is expressed by lymphoid and nonlymphoid cells, including B cell lymphoma lines. In this study, we have further explored IL-15Rα-mediated signaling in activated primary B cells and in Raji cells, a human B-lymphoblastoid cell line which expresses the IL-15Rα and IL-2Rγ chains, but lacks the IL-2Rβ chain. Stimulation of Raji cells with IL-15 induces their proliferation and rescues them from C2-ceramide-induced apoptosis. By immunoprecipitation and Western blotting, we show that treatment of Raji cells and activated primary B cells with IL-15 induces coprecipitation of Syk kinase with the IL-15Rα chain. Upon association, the activated Syk kinase phosphorylates the IL-15Rα chain as well as phospholipase Cγ, which coprecipitates with Syk. Furthermore, transfection of Raji cells with stem-loop Syk antisense oligonucleotides prevents IL-15Rα and phospholipase Cγ phosphorylation as well as the inhibition of apoptosis by IL-15. Mutation of a defined region of the intracellular signaling portion of IL-15Rα (Tyr227) abrogates both the IL-15Rα/Syk association and IL-15Rα phosphorylation. Taken together, this suggests that Syk kinase physically and functionally associates with the IL-15Rα chain in B cells and that Syk plays a key role in mediating IL-15-induced signal transduction, thus accounting for the distinct functional consequences of IL-15 vs IL-2 binding to B cells.
The Impact of H2-DM on Humoral Immune Responses
Authors: Alfonso Christopher , Han Jung-Ok , Williams G. Stuart , Karlsson Lars
Published: April 2014

H2-DM (DM, previously H2-M) facilitates the exchange of peptides bound to MHC class II molecules. In this study, we have used H2-DM-deficient (DM−/−) mice to analyze the influence of DM in the priming of B cell responses in vivo and for Ag presentation by B cells in vitro. After immunization, IgG Abs could be raised to a T-dependent Ag, 4-hydroxy-5-nitrophenylacetyl-OVA, in DM−/− mice, but closer analysis revealed the IgG response to be slower, diminished in titer, and composed of low-affinity Abs. The Ab response correlated with a vast reduction in the number of germinal centers in the spleen. The presentation of multiple epitopes by H2-Ab from distinct Ags was found to be almost exclusively DM-dependent whether B cells internalized Ags via fluid phase uptake or using membrane Ig receptors. The poor B cell response in vivo could be largely, but not completely restored by expression of a H2-Ead transgene, despite the fact that Ag presentation by H2-Ed/b molecules was found to be highly DM dependent. Hence, while substantial Ab responses can be raised in the absence of DM, this molecule is a crucial factor both for Ag processing and for the normal maturation of T-dependent humoral immune responses in vivo.
Surrogate Light Chain-Mediated Interaction of a Soluble Pre-B Cell Receptor with Adherent Cell Lines
Authors: Bradl Harald , Jäck Hans-Martin
Published: April 2014

Signals initiated by the precursor B cell receptor (pre-BCR) are critical for B cell progenitors to mature into precursor B cells. The pre-BCR consists of a homodimer of μH chains, the covalently associated surrogate L (SL) chain composed of VpreB and λ5, and the transmembrane signal molecules Igα and Igβ. One way to explain how maturation signals are initiated in late progenitor B cells is that the pre-BCR is transported to the cell surface and interacts from there with a ligand on stroma cells. To address this hypothesis, we first produced soluble Fab-like pre-BCR and BCR fragments, as well as SL chain, in baculovirus-infected insect cells. Flow cytometry revealed that, in contrast to Fab-like BCR fragments, the soluble pre-BCR binds to the surface of stroma and several other adherent cell lines, but not to B and T lymphoid suspension cells. The specific binding of the soluble pre-BCR to stroma cells is saturable, sensitive to trypsin digestion, and not dependent on bivalent cations. The binding of pre-BCR seems to be independent of the H chain of IgM (μH chain), because SL chain alone was able to interact with stroma cells. Finally, soluble pre-BCR specifically precipitated a 135-kDa protein from ST2 cells. These findings not only demonstrate for the first time the capacity of a pre-BCR to specifically bind to a structure on the surface of adherent cells, but also suggest that the pre-BCR interacts via its SL chain with a putative ligand on stroma cells.
Tumor-Specific Tc1, But Not Tc2, Cells Deliver Protective Antitumor Immunity
Authors: Kemp Roslyn A. , Ronchese Franca
Published: April 2014

We investigated whether secretion of multiple cytokines by CD8+ T cells is associated with improved protection against tumor challenge. We show that antitumor immunity induced by immunization with dendritic cells and a MHC class I-binding tumor peptide are dependent on secretion of IFN-γ but not IL-4 or IL-5 by host cells. To further address the role of IL-4 and IL-5 in antitumor immunity, tumor-specific TCR-transgenic CD8+ T cells were activated in vitro to generate cytotoxic T (Tc) 1 cells that secrete high IFN-γ and no IL-4 or IL-5 or Tc2 cells that secrete IL-4, IL-5, and some IFN-γ. Both cell types killed target cells in vitro. Tc1 and Tc2 cells were adoptively transferred into syngeneic hosts, and their ability to protect against tumor challenge was compared. Tc1 cells were able to significantly delay tumor growth, whereas Tc2 cells or Tc2 cells from IFN-γ−/− donors had no effect. This was due to neither the inability of Tc2 cells to survive in vivo or to migrate to the tumor site nor their inability to secrete IL-4 and/or IL-5 in the presence of limiting amounts of anti-CD3. However, IFN-γ secretion by Tc2 cells was triggered inefficiently by restimulation with Ag compared with anti-CD3. We conclude that the ability to secrete “type 2” cytokines, and cytotoxic ability, have a limited role in antitumor immune responses mediated by CD8+ T cells, whereas the capacity to secrete high amounts of IFN-γ remains the most critical antitumor effector mechanism in vivo.
Liver Damage by Infiltrating CD8+ T Cells Is Fas Dependent
Authors: Kennedy Norman J. , Russell Jennifer Q. , Michail Nina , Budd Ralph C.
Published: April 2014

Ag stimulation of CD8+ lymphocytes in vivo results in their migration to various tissues as well as the activation of a cytolytic program involving perforin, TNF-α, and Fas ligand. The liver is one of the main sites for infiltration by activated CD8+ T cells, and this is followed by the death of hepatocytes. The contribution of the various cytolytic components to this process is unclear. Hepatocyte damage by CD8+ T cells was studied using the MHC class I-restricted OVA-specific TCR transgenic mouse (OT-1) to examine the contribution of Fas to hepatocyte death. Activated CD8+ T cells from both OT-1 and Fas-deficient OT-1lpr mice migrated to the liver in similar numbers after OVA administration, but only in OT-1 mice was there evidence of significant hepatocyte damage histologically and by elevation of serum aspartate transaminase. These differences were not the result of inefficient induction of cytolytic activity in OT-1lpr liver T cells, since they were as cytolytic in vitro as OT-1 liver T cells. This was supported by findings of similar high levels of message for perforin, TNF-α, and Fas ligand in liver lymphocytes from both mice. These findings demonstrate that following Ag activation, infiltrating liver CD8+ T lymphocytes induce hepatocyte damage in a Fas-dependent manner.
Cutting Edge: Inhibition of Hepatitis B Virus Replication by Activated NK T Cells Does Not Require Inflammatory Cell Recruitment to the Liver
Authors: Kakimi Kazuhiro , Lane Thomas E. , Chisari Francis V. , Guidotti Luca G.
Published: April 2014

We have previously reported that intrahepatic NK T cells activated by α-galactosylceramide inhibit hepatitis B virus replication noncytopathically in the liver of transgenic mice. This effect is mediated by antiviral cytokines directly produced by activated NK T cells and/or by other cytokine-producing inflammatory cells that are recruited into the liver. In this study, we demonstrated that IFN-γ produced by activated NK T cells induced parenchymal and nonparenchymal cells of the liver to produce high levels of CXC chemokine ligands 9 and 10, which mediated the intrahepatic recruitment of lymphomononuclear inflammatory cells. Recruitment of these cells was not necessary for the antiviral activity, indicating that direct activation of the intrahepatic resident NK T cell is sufficient to control viral replication in this model.
CD46/CD3 Costimulation Induces Morphological Changes of Human T Cells and Activation of Vav, Rac, and Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase
Authors: Zaffran Yona , Destaing Olivier , Roux Agnès , Ory Stéphane , Nheu Thao , Jurdic Pierre , Rabourdin-Combe Chantal , Astier Anne L.
Published: April 2014

Efficient T cell activation requires at least two signals, one mediated by the engagement of the TCR-CD3 complex and another one mediated by a costimulatory molecule. We recently showed that CD46, a complement regulatory receptor for C3b as well as a receptor for several pathogens, could act as a potent costimulatory molecule for human T cells, highly promoting T cell proliferation. Indeed, we show in this study that CD46/CD3 costimulation induces a synergistic activation of extracellular signal-related kinase mitogen-activated protein kinase. Furthermore, whereas T lymphocytes primarily circulate within the bloodstream, activation may induce their migration toward secondary lymphoid organs or other tissues to encounter APCs or target cells. In this study, we show that CD46/CD3 costimulation also induces drastic morphological changes of primary human T cells, as well as actin relocalization. Moreover, we show that the GTP/GDP exchange factor Vav is phosphorylated upon CD46 stimulation alone, and that CD46/CD3 costimulation induces a synergistic increase of Vav phosphorylation. These results prompted us to investigate whether CD46/CD3 costimulation induced the activation of GTPases from the Rho family. Indeed, we report that the small GTPase Rac is also activated upon CD46/CD3 costimulation, whereas no change of Rho and Cdc42 activity could be detected. Therefore, CD46 costimulation profoundly affects T cell behavior, and these results provide important data concerning the biology of primary human T cells.
Tolerance to Nickel: Oral Nickel Administration Induces a High Frequency of Anergic T Cells with Persistent Suppressor Activity
Authors: Artik Suzan , Haarhuis Karin , Wu Xianzhu , Begerow Jutta , Gleichmann Ernst
Published: April 2014

We adapted our mouse model of allergic contact hypersensitivity to nickel for the study of tolerance. Sensitization in this model is achieved by the administration of nickel ions with H2O2; nickel ions alone are unable to prime naive T cells, but can restimulate primed ones. A 4-wk course of oral or i.p. administration of 10 mM NiCl2 to naive mice induced tolerance, preventing the induction of hypersensitivity for at least 20 wk; long term desensitization of nickel-sensitized mice, however, required continuous NiCl2 administration. When splenic T cells of orally tolerized donors, even after a treatment-free interval of 20 wk, were transferred to naive recipients, as with lymph node cells (LNC), they specifically prevented sensitization of the recipients. The LNC of such donors were anergic, because upon in vivo sensitization with NiCl2 in H2O2 and in vitro restimulation with NiCl2, they failed to show the enhanced proliferation and IL-2 production as seen with LNC of mice not tolerized before sensitization. As few as 102 bulk T cells, consisting of both CD4+ and CD8+ cells, were able to specifically transfer tolerance to nickel. A hypothesis is provided to account for this extraordinarily high frequency of nickel-reactive, suppressive T cells; it takes into account that nickel ions fail to act as classical haptens, but form versatile, unstable metal-protein and metal-peptide complexes. Furthermore, a powerful amplification mechanism, such as infectious tolerance, must operate which allows but a few donor T cells to tolerize the recipient.
IL-2, -4, and -15 Differentially RegulateO-Glycan Branching and P-Selectin Ligand Formation in Activated CD8 T Cells
Authors: Carlow Douglas A. , Corbel Stéphane Y. , Williams Michael J. , Ziltener Hermann J.
Published: April 2014

The glycosyltransferase core 2 β1–6 N-acetylglucosaminyl transferase (C2GnT1 or C2GlcNAcT1) is responsible for formation of branched structures on O-glycans present on cell surface glycoproteins. The O-glycan branch created by C2GnT1 is physiologically important insofar as only this structure can be extended and modified to yield P-selectin ligands that promote initial interactions between extravasating lymphocytes and endothelia. In mature T cells, C2GnT1 activity is thought to be induced as an intrinsic consequence of T cell activation. Through analysis of C2GnT1-dependent epitopes on CD43 and CD45RB we have found that in activated CD8+ T cells expression of C2GnT1 was dependent upon exposure to specific cytokines rather than being induced as a direct consequence of activation. Activated CD8+ cells became receptive to strong induction of C2GnT1 expression and P-selectin ligand expression in response to IL-2, moderate induction by IL-15, and minimal induction in response to IL-4. Our observations clarify the relationship between T cell activation and C2GnT1 expression, demonstrate the differential impact of distinct cytokines on expression of C2GnT1 activity and P-selectin ligand, and reinforce the concept that the cytokine milieu subsequent to activation can influence adhesion systems that dictate lymphocyte homing properties.
NFκB and Sp1 Elements Are Necessary for Maximal Transcription of Toll-like Receptor 2 Induced byMycobacterium avium
Authors: Wang Tianyi , Lafuse William P. , Zwilling Bruce S.
Published: April 2014

We have previously reported that Toll-like receptor (TLR) 2 mRNA was induced after infection with Mycobacterium avium. To investigate the molecular basis of TLR2 expression in macrophages, we cloned and analyzed the murine putative 5′-proximal promoter. Transient transfection of a 326-bp region from nucleotides −294-+32 relative to the first transcription start site was sufficient to induce maximal luciferase activity at the basal level and after infection with M. avium in J774A.1 cells. Sequence analysis showed that the region lacked a TATA box but contained two typical stimulating factor (Sp) 1 sites, two NF-κB sites, one IFN-regulatory factor site and one AP-1 site. Site-directed mutagenesis revealed that the NF-κB and Sp1 sites but not the IFN-regulatory factor site or the AP-1 site contributed to the basal level and the induction of luciferase activity during M. avium infection. Binding of Sp1/Sp3 and NF-κB (p50/p65) was confirmed by EMSA. Further studies showed that three copies of Sp1 elements or NF-κB elements are not sufficient to confer M. avium induction on a heterologous promoter. By contrast, overexpression of NF-κB p65 caused a strong increase in transcription from an intact TLR2 promoter, whereas it caused only a partial increase in promoter activity when cotransfected with the TLR2 promoter with one of the Sp1 sites mutated. Sp1 and NF-κB were the minimum mammalian transcription factors required for effective TLR2 transcriptional activity when transfected into Drosophila Schneider cells. Together, these data provide genetic and biochemical evidence for NF-κB as well as Sp1 in regulating TLR2 transcription.
Unusual Germline DSP2 Gene Accounts for All Apparent V-D-D-J Rearrangements in Newborn, But Not Adult, MRL Mice
Authors: Kompfner Elizabeth , Oliveira Paula , Montalbano Alina , Feeney Ann J.
Published: April 2014

Anti-dsDNA autoantibodies in MRL mice contain a higher than average frequency of atypical complementarity-determining regions 3, including those made with D-D rearrangements. It has been reported that MRL mice have an intrinsically high frequency of creating VDDJ rearrangements; however, we show in this study that the majority of these apparent D-D rearrangements in B cell progenitors can be accounted for by a very novel germline DH gene in mice of the Ighj haplotype. This gene has the appearance of a D to D rearrangement due to the duplication of 9 bp common to most DSP2 genes. Germline DSP2 genes from Ighj mice were amplified, cloned, and sequenced, showing the presence of this novel gene as well as a new allele of a conventional DSP2 gene. Sequencing of D-J rearrangements revealed that Ighj mice also have a different allele of DFL16.1 and apparently lack DFL16.2. Despite the existence of this new DSP gene, analysis of VDJ rearrangements from adult bone marrow pre-B cells of MRL/lpr mice still revealed the presence of complementarity-determining region 3 containing apparent D-D joinings in 4.6% of the sequences. C3H pre-B cells had 4.2% of sequences with apparent VDDJ rearrangements, and BALB/c pre-B cells had ∼2%. DDJ intermediates were also observed, but at a lower frequency. However, strikingly, no VDDJ rearrangements were observed in newborn sequences, suggesting the process of assembly of VDJ rearrangements is fundamentally different in newborn mice vs adult mice.
A Protective and Agonistic Function of IL-12p40 in Mycobacterial Infection
Authors: Hölscher Christoph , Atkinson Robert A. , Arendse Berenice , Brown Najmeeyah , Myburgh Elmarie , Alber Gottfried , Brombacher Frank
Published: April 2014

IL-12p35−/−p40−/− mice are highly susceptible to Mycobacterium bovis bacillus Calmette-Guérin (BCG) or Mycobacterium tuberculosis infection. In this study IL-12p35−/− mice, which are able to produce endogenous IL-12p40, cleared M. bovis BCG and showed reduced susceptibility to pulmonary M. tuberculosis infection, which was in striking contrast to the outcome of mycobacterial infection in IL-12p35−/−p40−/− mice. Resistance in wild-type and IL-12p35−/− mice was accompanied by protective granuloma formation and Ag-specific delayed-type hypersensitivity responses, which were impaired in susceptible IL-12p35−/− p40−/− mice. Furthermore, IL-12p35−/− mice, but not IL-12p35−/−p40−/− mice, mounted Ag-specific Th1 and cytotoxic T cell responses. In vivo therapy with rIL-12p40 homodimer restored the impaired delayed-type hypersensitivity responses in M. bovis BCG-infected IL-12p35−/−p40−/− mice and reverted them to a more resistant phenotype. Together, these results show evidence for a protective and agonistic role of endogenous and exogenous IL-12p40 in mycobacterial infection, which is independent of IL-12p70.
The Role of Antigen in the Localization of Naive, Acutely Activated, and Memory CD8+ T Cells to the Lung During Influenza Pneumonia
Authors: Topham David J. , Castrucci Maria R. , Wingo F. Suzette , Belz Gabrielle T. , Doherty Peter C.
Published: April 2014

The role of Ag in the recruitment and localization of naive, acutely activated, and memory CD8+ T cells to the lung during influenza infection was explored using TCR-transgenic (Tg) mice. Naive, Thy1.2+CD8+ OT-I TCR-Tg cells were primed and recruited to the lung after transfer into congenic Thy1.1+ recipients challenged with a genetically engineered influenza virus (influenza A/WSN/33 (WSN)-OVAI) containing the Kb restricted OVA257–264 epitope (siinfekl) in the viral neuraminidase stalk. However, if the transferred animals were infected with a similar influenza virus that expressed an irrelevant Kb epitope (WSN-PEPII), no TCR-Tg T cells were detectable in the lung, although they were easily visible in the lymphoid organs. Conversely, there were substantial numbers of OT-I cells found in the lungs of WSN-PEPII-infected mice when the animals had been previously, or were concurrently, infected with a recombinant vaccinia virus expressing OVA. Similar results were obtained with nontransgenic populations of memory CD8+ T cells reactive to a murine γ-herpesvirus-68 Ag. Interestingly, the primary host response to the immunodominant influenza nucleoprotein epitope was not affected by the presence of memory or recently activated OT-I T cells. Thus, although Ag is required to activate the T cells, the subsequent localization of T cells to the lung during a virus infection is a property of recently activated and memory T cells and is not necessarily driven by Ag in the lung.
Stability of Surface H-2Kb, H-2Db, and Peptide-Receptive H-2Kb on Splenocytes
Authors: Su Ruey-Chyi , Miller Richard G.
Published: April 2014

We have used flow cytometry to study the stability and peptide-binding capability of MHC class I (MHC-I) on the surface of normal C57BL/6 mouse T lymphoblasts. The MHC-I molecules on each cell are nearly evenly divided into two populations with mean half-life values of ∼1 and 20 h. Our observations suggest that members of the later contain peptide bound with medium to high affinity. Cell surface MHC-I molecules capable of binding exogenous peptide (thus, “peptide-receptive”) belong almost entirely to the less stable population. Before exogenous peptide can bind, MHC-I must undergo a change, probably loss of a very low affinity peptide. For MHC-I-Kb, we found that the maximum rate for binding of exogenous peptide corresponds to a t1/2 value of 12 min. To maintain the 50:50 steady-state distribution of long- vs short-lived MHC-I molecules on the cell surface, ∼20 short-lived molecules must be exported to the cell surface for each long-lived molecule.
Divergent Therapeutic and Immunologic Effects of Oligodeoxynucleotides with Distinct CpG Motifs
Authors: Ballas Zuhair K. , Krieg Arthur M. , Warren Thomas , Rasmussen Wendy , Davis Heather L. , Waldschmidt Marinella , Weiner George J.
Published: April 2014

Immune stimulatory oligodeoxynucleotides (ODN) with unmethylated CpG motifs are potent inducers of both innate and adaptive immunity. It initially appeared that a single type of optimal CpG motif would work in all applications. We now report that specific motifs of CpG ODN can vary dramatically in their ability to induce individual immune effects and that these differences impact on their antitumor activity in different tumor models. In particular, a distinct type of CpG motif, which has a chimeric backbone in combination with poly(G) tails, is a potent inducer of NK lytic activity but has little effect on cytokine secretion or B cell proliferation. One such NK-optimized CpG ODN (1585) can induce regression of established melanomas in mice. Surprisingly, no such therapeutic effects were seen with CpG ODN optimized for activation of B cells and Th1-like cytokine expression (ODN 1826). The therapeutic effects of CpG 1585 in melanoma required the presence of NK but not T or B cells and were not associated with the induction of a tumor-specific memory response. In contrast, CpG 1826, but not CpG 1585, was effective at inducing regression of the EL4 murine lymphoma; this rejection was associated with the induction of a memory response and although NK cells were necessary, they were not sufficient. These results demonstrate that selection of optimal CpG ODN for cancer immunotherapy depends upon a careful analysis of the cellular specificities of various CpG motifs and an understanding of the cellular mechanisms responsible for the antitumor activity in a particular tumor.
c-Rel Is Required for the Protection of B Cells from Antigen Receptor-Mediated, But Not Fas-Mediated, Apoptosis
Authors: Owyang Alexander M. , Tumang Joseph R. , Schram Brian R. , Hsia Constance Y. , Behrens Timothy W. , Rothstein Thomas L. , Liou Hsiou-Chi
Published: April 2014

The NF-κB/Rel transcription factor family has been shown to protect many cell types from apoptotic signals. However, it is not known whether NF-κB is required for all survival pathways and whether each NF-κB member plays a unique or a redundant role. Here we describe the results of studies on the role of c-Rel in survival. Mature B cells from c-Rel−/− mice exhibit defects in survival, including sensitivity to Ag receptor-mediated apoptosis as well as increased sensitivity to ionizing radiation and glucocorticoids. Transgene expression of Bcl-xL, a c-Rel target gene, rescues c-Rel−/− B cells from their survival defects. Thus, c-Rel-dependent survival pathways are crucial for protection from apoptotic signals that target the mitochondrial pathway. Despite a lack of Bcl-xL, c-Rel−/− B cells can still be rescued from Fas-mediated apoptosis via B cell receptor signaling. The Fas apoptosis inhibitor molecule and FLICE inhibitory protein (c-FLIP) proteins are up-regulated normally in c-Rel−/− B cells, and these two molecules may play a more physiological role in the Fas pathway. Furthermore, unlike the TNF sensitivity of RelA−/− fibroblasts, c-Rel-deficient fibroblasts are refractory to TNF-mediated cell death. Thus, c-Rel is dispensable for protection against death receptor-mediated apoptosis. Taken together, our data suggest that distinct NF-κB/Rel members are required for protecting cells from different types of apoptotic signals.
Characterization of Virus-Mediated Inhibition of Mixed Chimerism and Allospecific Tolerance
Authors: Williams Matthew A. , Tan Joyce T. , Adams Andrew B. , Durham Megan M. , Shirasugi Nozomu , Whitmire Jason K. , Harrington Laurie E. , Ahmed Rafi , Pearson Thomas C. , Larsen Christian P.
Published: April 2014

Simultaneous blockade of the CD28 and CD40 T cell costimulatory pathways has been shown to effectively promote skin allograft survival in mice. Furthermore, blockade of one or both of these pathways has played a central role in the development of strategies to induce mixed hematopoietic chimerism and allospecific tolerance. It has recently been observed that the beneficial effects of CD40 blockade and donor splenocytes in prolonging skin graft survival can be abrogated by some viral infections, including lymphocytic choriomeningitis virus (LCMV). In this study, we show that LCMV infection prevents prolonged allograft survival following CD28/CD40 combined blockade. We further show that LCMV prevents the induction of allospecific tolerance and mixed hematopoietic chimerism, while delay of infection for 3–4 wk posttransplant has no effect on tolerance induction. Because of reports of anti-H-2d activity following LCMV infection, we assayed the ability of LCMV-specific T cells to respond to alloantigen at a single cell level. Although we confirm that LCMV infection induces the generation of alloreactive cells, we also demonstrate that LCMV-specific T cells do not divide in response to alloantigen. The alloresponse suppressed by costimulation blockade is restored by LCMV infection and correlates with increased dendritic cell maturation. We hypothesize that the costimulation blockade-resistant rejection mediated by LCMV could be partly attributable to the up-regulation of alternative costimulatory pathways subsequent to LCMV-induced dendritic cell maturation.