bluuebunny/crossref_metadata_2025 huggingface dataset:
Superantigen-Induced TCR α Locus Secondary Rearrangement: Role in Tolerance Induction
Authors: Huang Ching-Yu , Golub Rachel , Wu Gillian E. , Kanagawa Osami
Published: April 2014

Immunization with superantigen in vivo induces transient activation of superantigen-specific T cells, followed by a superantigen-nonresponsive state. In this study, using a TCR α knock-in mouse in which the knock-in α-chain can be replaced with endogenous α-chain through secondary rearrangement, we show that immunization of superantigen changes the TCR α-chain expression on peripheral superantigen-specific T cells, induces expression of recombination-activating genes, and generates DNA double-strand breaks at the TCR α-chain locus. These results suggest that viral superantigens are capable of inducing peripheral TCR revision. Our findings thus provide a new perspective on pathogen-immune system interaction.
Genetically Modified HLA Class I Molecules Able to Inhibit Human NK Cells Without Provoking Alloreactive CD8+ CTLs
Authors: Sharland Alexandra , Patel Amy , Lee Josie Han , Cestra Aimee E. , Saidman Susan , Waneck Gerald L.
Published: April 2014

Human NK cells are likely to be important effectors of xenograft rejection. Expression of HLA class I molecules by transfected porcine cells can protect them from human NK cell-mediated lysis; however, this strategy has the potential to augment the anti-graft response by recipient CD8+ T cells recognizing foreign pig peptides presented by HLA. In this study we show that the introduction of a mutation (D227K) in the α3 domain of HLA-Cw3 abrogates its recognition by CD8-dependent T cells but leaves intact its ability to function as an inhibitory ligand for NK cells. Such genetically modified molecules may have potential therapeutic applications in the prevention of delayed xenograft rejection and in the facilitation of allogeneic and xenogeneic bone marrow engraftment.
Contribution of Langerhans Cell-Derived IL-18 to Contact Hypersensitivity
Authors: Wang Binghe , Feliciani Claudio , Howell Brandon G. , Freed Irwin , Cai Qinchao , Watanabe Hideaki , Sauder Daniel N.
Published: April 2014

The epidermal Langerhans cells (LC), a member of the dendritic cell family, and the LC-derived cytokine IL-12 play a pivotal role in the initiation of contact hypersensitivity (CHS), a Th1 immune response in the skin. Because IL-18, another LC-derived cytokine, shares functional and biological properties with IL-12, we examined a potential role for IL-18 in CHS initiation. Our studies demonstrated that during the induction phase of murine CHS, IL-18 mRNA was significantly up-regulated in the skin-draining lymph nodes (LN). Migratory hapten-modified LC in LN expressed high levels of IL-18 mRNA and secreted functional IL-18 protein. LN cells produced significant amounts of IFN-γ following in vitro IL-12 stimulation, which could be partially blocked by anti-IL-18 Ab, suggesting a synergistic role for endogenous IL-18 in IFN-γ production by LN cells. Because mature IL-18 requires cleavage of immature precursors by caspase-1, we further examined IL-12-induced IFN-γ production in caspase-1−/− LN cells. An impaired IFN-γ production was seen in caspase-1−/− LN cells, which could be restored by addition of exogenous IL-18, supporting a role for caspase-1-cleaved, mature IL-18 in IFN-γ production. Finally, in vivo studies showed that CHS responses were significantly inhibited in mice treated with neutralizing IL-18 Ab as well as in caspase-1−/− mice deficient in mature IL-18, indicating functional relevance for IL-18 in CHS. Taken together, our studies demonstrate that LC-derived IL-18 significantly contributes to CHS initiation.
DQ 65–79, a Peptide Derived from HLA Class II, Induces IκB Expression
Authors: Jiang Yun , Chen Daniel , Lyu Shu-Chen , Ling Xuefeng , Krensky Alan M. , Clayberger Carol
Published: April 2014

A synthetic peptide corresponding to residues 65–79 of the α helix of the α-chain of the class II HLA molecule DQA03011 (DQ 65–79) inhibits the proliferation of human T lymphocytes in an allele nonrestricted manner. By using microarray technology, we found that expression of 29 genes was increased or decreased in a human CTL cell line after treatment with DQ 65–79. This study focuses on one of these genes, IκB-α, whose expression is increased by DQ 65–79. IκB proteins, including IκB-α and IκB-β, are increased in T cells treated with DQ 65–79. Nuclear translocation of the NF-κB subunits p65 and p50 is decreased in T cells after treatment with DQ 65–79, while elevated levels of p65 and p50 are present in cytosol. DQ 65–79 inhibits the degradation of IκB-α mRNA and inhibits the activity of IκB kinase. These findings indicate that the DQ 65–79 peptide increases the level of IκB proteins, thereby preventing nuclear translocation of the transcription factor, NF-κB, and inhibiting T cell proliferation.
Yin Yang 1, Oct1, and NFAT-4 Form Repeating, Cyclosporin-Sensitive Regulatory Modules Within the Murine CD21 Intronic Control Region
Authors: Zabel Mark D. , Wheeler Wells , Weis Janis J. , Weis John H.
Published: April 2014

The murine complement receptor type 2 gene (Cr2/CD21) is expressed by murine B and follicular dendritic cells, but not murine T cells. We have previously shown that appropriate transcriptional control of the CD21 gene requires the CD21 promoter as well as intronic sequences. We have also demonstrated that altering chromatin structure by inhibiting histone deacetylases induces CD21 expression in murine T cells by increasing the accessibility of promoter and intronic regulatory elements. In this report, we identify seven distinct regulatory areas within the first intron of the murine CD21 gene that are conserved between mouse and human CD21 intronic sequences. EMSA competition and supershift analyses reveal the formation of multiple DNA-protein complexes at these sites that include Yin Yang 1, Oct1, and NFAT-4. NFAT-containing complexes were altered in B cells treated with the NFAT inhibitor cyclosporin A and correlated with a repression of CD21 gene transcription implicating NFAT transcriptional control. Functional data revealed that no single region conferred cell-specific reporter gene expression, but rather the entire CD21 regulatory element was required to confer cell-specific gene expression. Taken together, these data demonstrate the formation of repeating, overlapping regulatory modules, all of which are required to coordinately control the cell-specific expression of the murine CD21 gene. We propose a model in which Yin Yang 1 and Oct1 may recruit histone deacetylase to multiple sites in the CD21 intronic regulatory element in nonexpressing cells and NFAT either displaces this histone deacetylase or recruits a histone acetylase to allow the formation of a functional transcriptional complex in expressing cells.
The Rac2 Guanosine Triphosphatase Regulates B Lymphocyte Antigen Receptor Responses and Chemotaxis and Is Required for Establishment of B-1a and Marginal Zone B Lymphocytes
Authors: Croker Ben A. , Tarlinton David M. , Cluse Leonie A. , Tuxen Alana J. , Light Amanda , Yang Feng-Chun , Williams David A. , Roberts Andrew W.
Published: April 2014

We have defined roles for the hemopoietic-specific Rho guanosine triphosphatase, Rac2, in B lymphocyte development and function through examination of rac2−/− mice. Rac2-deficient mice displayed peripheral blood B lymphocytosis and marked reductions in peritoneal cavity B-1a lymphocytes, marginal zone B lymphocytes, and IgM-secreting plasma cells as well as reduced concentrations of serum IgM and IgA. The rac2−/− B lymphocytes exhibited reduced calcium flux following coligation of B cell AgR and CD19 and reduced chemotaxis in chemokine gradients. T cell-independent responses to DNP-dextran were of reduced magnitude, but normal kinetics, in rac2−/− mice, while T-dependent responses to nitrophenyl-keyhole limpet hemocyanin were subtly abnormal. Rac2 is therefore an essential element in regulating B lymphocyte functions and maintaining B lymphocyte populations in vivo.
Transmembrane TNF Induces an Efficient Cell-Mediated Immunity and Resistance toMycobacterium bovisBacillus Calmette-Guérin Infection in the Absence of Secreted TNF and Lymphotoxin-α
Authors: Olleros Maria L. , Guler Reto , Corazza Nadia , Vesin Dominique , Eugster Hans-Pietro , Marchal Gilles , Chavarot Pierre , Mueller Christoph , Garcia Irene
Published: April 2014

The contribution of a transmembrane (Tm) form of TNF to protective immunity against Mycobacterium bovis bacillus Calmette-Guérin (BCG) was studied in transgenic (tg) mice expressing a noncleavable Tm TNF but lacking the TNF/lymphotoxin-α (LT-α) locus (Tm TNF tg mice). These mice were as resistant to BCG infection as wild-type mice, whereas TNF/LT-α−/−, TNF−/−, and LT-α−/− mice succumbed. Tm TNF tg mice developed granulomas of smaller size but at 2- to 4-fold increased frequencies compared with wild-type mice. Granulomas were mainly formed by monocytes and activated macrophages expressing Tm TNF mRNA and accumulating acid phosphatase. NO synthase 2 activation as a key macrophage bactericidal mechanism was low during the acute phase of infection in Tm TNF tg mice but was still sufficient to limit bacterial growth and increased in late infection. While infection with virulent Mycobacterium tuberculosis resulted in very rapid death of TNF/LT-α−/− mice, it also resulted in survival of Tm TNF tg mice which presented an increase in the number of CFU in spleen (5-fold) and lungs (10-fold) as compared with bacterial load of wild-type mice. In conclusion, the Tm form of TNF induces an efficient cell-mediated immunity and total resistance against BCG even in the absence of LT-α and secreted TNF. However, Tm TNF-mediated protection against virulent M. tuberculosis infection can also be efficient but not as strong as in BCG infection, in which cognate cellular interactions may play a more predominant role in providing long-term surveillance and containment of BCG-infected macrophages.
Preferential Infection of Immature Dendritic Cells and B Cells by Mouse Mammary Tumor Virus
Authors: Vacheron Sonia , Luther Sanjiv A. , Acha-Orbea Hans
Published: April 2014

Until now it was thought that the retrovirus mouse mammary tumor virus preferentially infects B cells, which thereafter proliferate and differentiate due to superantigen-mediated T cell help. We describe in this study that dendritic cells are infectable at levels comparable to B cells in the first days after virus injection. Moreover, IgM knockout mice have chronically deleted superantigen-reactive T cells after MMTV injection, indicating that superantigen presentation by dendritic cells is sufficient for T cell deletion. In both subsets initially only few cells were infected, but there was an exponential increase in numbers of infected B cells due to superantigen-mediated T cell help, explaining that at the peak of the response infection is almost exclusively found in B cells. The level of infection in vivo was below 1 in 1000 dendritic cells or B cells. Infection levels in freshly isolated dendritic cells from spleen, Langerhans cells from skin, or bone marrow-derived dendritic cells were compared in an in vitro infection assay. Immature dendritic cells such as Langerhans cells or bone marrow-derived dendritic cells were infected 10- to 30-fold more efficiently than mature splenic dendritic cells. Bone marrow-derived dendritic cells carrying an endogenous mouse mammary tumor virus superantigen were highly efficient at inducing a superantigen response in vivo. These results highlight the importance of professional APC and efficient T cell priming for the establishment of a persistent infection by mouse mammary tumor virus.
Reduced Functional Capacity of CD8+ T Cells Expanded by Post-Exposure Vaccination of γ-Herpesvirus-Infected CD4-Deficient Mice
Authors: Liu Haiyan , Andreansky Samita , Diaz Gabriela , Hogg Twala , Doherty Peter C.
Published: April 2014

Mice (I-Ab−/−) that lack CD4+ T cells remain healthy for at least three months after respiratory exposure to the murine γ-herpesvirus 68 (γHV68), then succumb with symptoms of chronic wasting disease. Postexposure challenge of γHV68-infected I-Ab+/+ and I-Ab−/− mice with a recombinant vaccinia virus (Vacc-p56) expressing an antigenic γHV68 peptide caused a massive increase in the numbers of Dbp56-specific CD8+ T cells. Previous experiments showed that, despite the large numbers of potential CTL effectors, there was little effect on the long-term survival of the CD4-deficient group and no diminution in the level of persistent virus shedding and latency. Comparison of the expanded CD8+Dbp56+ sets in the I-Ab+/+ and I-Ab−/− mice indicated that these two T cell populations were not identical. More CD69highCD8+ Dbp56+ T cells were found in the CD4-deficient mice, an effect that might be thought to reflect higher Ag load. By contrast, the mean fluorescence intensity of staining for the CD44 glycoprotein was diminished on CD8+Dbp56+ T cells from the I-Ab−/− group, the level of CTL activity was lower on a per cell basis, and the relative prevalence of IFN-γ+TNF-α+ T cells detected after in vitro stimulation with the p56 peptide was decreased. Given that this experimental system provides an accessible model for evaluating postexposure vaccination protocols that might be used in diseases like HIV/AIDS, the further need is to clarify the underlying molecular mechanisms and the relative significance of lack of CD4+ T help vs higher Ag load for these expanded CD8+ effector populations.
Tumor-Specific CTL Kill Murine Renal Cancer Cells Using Both Perforin and Fas Ligand-Mediated Lysis In Vitro, But Cause Tumor Regression In Vivo in the Absence of Perforin
Authors: Seki Naoko , Brooks Alan D. , Carter Clive R. D. , Back Timothy C. , Parsoneault Erin M. , Smyth Mark J. , Wiltrout Robert H. , Sayers Thomas J.
Published: April 2014

Kidney cancer is a devastating disease; however, biological therapies have achieved some limited success. The murine renal cancer Renca has been used as a model for developing new preclinical approaches to the treatment of renal cell carcinoma. Successful cytokine-based approaches require CD8+ T cells, but the exact mechanisms by which T cells mediate therapeutic benefit have not been completely identified. After successful biological therapy of Renca in BALB/c mice, we generated CTLs in vitro using mixed lymphocyte tumor cultures. These CTL mediated tumor-specific H-2Kd-restricted lysis and production of IFN-γ, TNF-α, and Fas ligand (FasL) in response to Renca. CTL used both granule- and FasL-mediated mechanisms to lyse Renca, although granule-mediated killing was the predominant lytic mechanism in vitro. The cytokines IFN-γ and TNF-α increased the sensitivity of Renca cells to CTL lysis by both granule- and FasL-mediated death pathways. Adoptive transfer of these anti-Renca CTL into tumor-bearing mice cured most mice of established experimental pulmonary metastases, and successfully treated mice were immune to tumor rechallenge. Interestingly, we were able to establish Renca-specific CTL from mice gene targeted for perforin (pfp−/−) mice. Although these pfp−/− CTL showed reduced cytotoxic activity against Renca, their IFN-γ production in the presence of Renca targets was equivalent to that of wild-type CTL, and adoptive transfer of pfp−/− CTL was as efficient as wild-type CTL in causing regression of established Renca pulmonary metastases. Therefore, although granule-mediated killing is of paramount importance for CTL-mediated lysis in vitro, some major in vivo effector mechanisms clearly are independent of perforin.
Depletion of CD8+ T Cells Exacerbates CD4+ Th Cell-Associated Inflammatory Lesions During Murine Mycoplasma Respiratory Disease
Authors: Jones Harlan P. , Tabor Leslie , Sun Xiangle , Woolard Matthew D. , Simecka Jerry W.
Published: April 2014

Mycoplasma infection is a leading cause of pneumonia worldwide and can lead to other respiratory complications. A component of mycoplasma respiratory diseases is immunopathologic, suggesting that lymphocyte activation is a key event in the progression of these chronic inflammatory diseases. The present study delineates the changes in T cell populations and their activation after mycoplasma infection and determines their association with the pathogenesis of murine Mycoplasma respiratory disease, due to Mycoplasma pulmonis infection. Increases in T cell population numbers in lungs and lower respiratory lymph nodes were associated with the development of mycoplasma respiratory disease. Although both pulmonary Th and CD8+ T cells increased after mycoplasma infection, there was a preferential expansion of Th cells. Mycoplasma-specific Th2 responses were dominant in lower respiratory lymph nodes, while Th1 responses predominated in spleen. However, both mycoplasma-specific Th1 and Th2 cytokine (IL-4 and IFN-γ) responses were present in the lungs, with Th1 cell activation as a major component of the pulmonary Th cell response. Although a smaller component of the T cell response, mycoplasma-specific CD8+ T cells were also a significant component of pulmonary lymphoid responses. In vivo depletion of CD8+ T cells resulted in dramatically more severe pulmonary disease, while depletion of CD4+ T cells reduced its severity, but there was no change in mycoplasma numbers in lungs after cell depletion. Thus, mycoplasma-specific Th1 and CD8+ T cell activation in the lung plays a critical regulatory role in development of immunopathologic reactions in Mycoplasma respiratory disease.
Activation of the Lectin Complement Pathway by H-Ficolin (Hakata Antigen)
Authors: Matsushita Misao , Kuraya Mikio , Hamasaki Naotaka , Tsujimura Mitsushi , Shiraki Hiroshi , Fujita Teizo
Published: April 2014

Ficolins are a group of proteins which consist of a collagen-like domain and a fibrinogen-like domain. In human serum, there are two types of ficolins named L-ficolin/P35 and H-ficolin (Hakata Ag), both of which have lectin activity. We recently demonstrated that L-ficolin/P35 is associated with mannose-binding lectin (MBL)-associated serine proteases (MASP) 1 and 2 and small MBL-associated protein (sMAP), and that the complex activates the lectin pathway. In this study, we report the characterization of H-ficolin in terms of its ability to activate complement. Western blotting analysis showed the presence of MASP-1, MASP-2, MASP-3, and sMAP in H-ficolin preparations isolated from Cohn Fraction III. The MASPs in the preparations had proteolytic activities against C4, C2, and C3 in the fluid phase. When H-ficolin preparations were bound to anti-H-ficolin Ab which had been coated on ELISA plates, they activated C4, although no C4 activation was noted when anti-MBL and anti-L-ficolin/P35 were used. H-ficolin binds to PSA, a polysaccharide produced by Aerococcus viridans. C4 was activated by H-ficolin preparations bound to PSA which had been coated on ELISA plates. These results indicate that H-ficolin is a second ficolin which is associated with MASPs and sMAP, and which activates the lectin pathway.
Urokinase Receptor Is Necessary for Adequate Host Defense Against Pneumococcal Pneumonia
Authors: Rijneveld Anita W. , Levi Marcel , Florquin Sandrine , Speelman Peter , Carmeliet Peter , van der Poll Tom
Published: April 2014

Cell recruitment is a multistep process regulated by cytokines, chemokines, and growth factors. Previous work has indicated that the urokinase plasminogen activator receptor (uPAR) may also play a role in this mechanism, presumably by an interaction with the β2 integrin CD11b/CD18. Indeed, an essential role of uPAR in neutrophil recruitment during pulmonary infection has been demonstrated for β2 integrin-dependent respiratory pathogens. We investigated the role of uPAR and urokinase plasminogen activator (uPA) during pneumonia caused by a β2 integrin-independent respiratory pathogen, Streptococcus pneumoniae. uPAR-deficient (uPAR−/−), uPA-deficient (uPA−/−), and wild-type (Wt) mice were intranasally inoculated with 105 CFU S. pneumoniae. uPAR−/− mice showed reduced granulocyte accumulation in alveoli and lungs when compared with Wt mice, which was associated with more S. pneumoniae CFU in lungs, enhanced dissemination of the infection, and a reduced survival. In contrast, uPA−/− mice showed enhanced host defense, with more neutrophil influx and less pneumococci in the lungs compared with Wt mice. These data suggest that uPAR is necessary for adequate recruitment of neutrophils into the alveoli and lungs during pneumonia caused by S. pneumoniae, a pathogen eliciting a β2 integrin-independent inflammatory response. This function is even more pronounced when uPAR is unoccupied by uPA.
Reduced Expression of Nuclear Cyclic Adenosine 5′-Monophospate Response Element-Binding Proteins and IFN-γ Promoter Function in Disease Due to an Intracellular Pathogen
Authors: Samten Buka , Ghosh Paritosh , Yi Ae-Kyung , Weis Stephen E. , Lakey David L. , Gonsky Rivkah , Pendurthi Usha , Wizel Benjamin , Zhang Yueru , Zhang Ming , Gong Jianhua , Fernandez Marilyn , Safi Hassan , Vankayalapati Ramakrishna , Young Howard A. , Barnes Peter F.
Published: April 2014

Mycobacterium tuberculosis-induced IFN-γ protein and mRNA expression have been shown to be reduced in tuberculosis patients, compared with healthy tuberculin reactors. To determine whether this decrease was associated with reduced activity of the IFN-γ promoter, we first studied binding of nuclear proteins to the radiolabeled proximal IFN-γ promoter (−71 to −40 bp), using EMSAs with nuclear extracts of freshly isolated peripheral blood T cells. Nuclear extracts of T cells from most tuberculosis patients showed markedly reduced expression of proteins that bind to the proximal IFN-γ promoter, compared with findings in nuclear extracts of T cells from healthy tuberculin reactors. These DNA-binding complexes contained CREB proteins, based on competitive EMSAs, supershift assays, and Western blotting with an anti-CREB Ab. Transient transfection of PBLs with a luciferase reporter construct under the control of the IFN-γ promoter revealed reduced IFN-γ promoter activity in tuberculosis patients. Transient transfection of Jurkat cells with a dominant-negative CREB repressor plasmid reduced IFN-γ promoter activity. These data suggest that reduced expression of CREB nuclear proteins in tuberculosis patients results in decreased IFN-γ promoter activity and reduced IFN-γ production.
Analysis of the Gene Expression Profile Activated by the CC Chemokine Ligand 5/RANTES and by Lipopolysaccharide in Human Monocytes
Authors: Locati Massimo , Deuschle Ullrich , Massardi Maria L. , Martinez Fernando O. , Sironi Marina , Sozzani Silvano , Bartfai Tamas , Mantovani Alberto
Published: April 2014

The gene expression profile induced by the CC chemokine ligand (CCL) 5/RANTES in human monocytes was examined using the oligonucleotide array technology. Of 5600 transcripts examined, 42 were consistently induced by CCL5, and none were suppressed. Chemokine-inducible transcripts could be clustered in functional groups, including selected cytokines and receptors (e.g., IL-1β, CCL2/monocyte chemotactic protein-1, and the CCL5 receptor CCR1) and molecules involved in extracellular matrix recognition and digestion (e.g., CD44 splice transcripts, urokinase-type plasminogen activator receptor, matrix metalloprotease (MMP)-9 , and MMP-19). Transcript expression, confirmed by quantitative real-time PCR analysis for selected genes, was associated with protein induction for some (e.g., CCL2), but not all (e.g., IL-1β), transcripts examined. The chemokine-induced gene profile was distinct from that activated by LPS, a prototypic phagocyte activator. Although certain transcripts were stimulated by both agonists (e.g., IL-1β and CCL2), others were induced only by either LPS (e.g., TNF-α and IL-6) or CCL5 (e.g., MMP-19) or were divergently regulated (e.g., CCR1). Thus, CCL5, a prototypic CC inflammatory chemokine, activates a restricted transcriptional program in monocytes distinct from that induced by the prototypic pathogen-derived proinflammatory stimulant LPS. Chemokine-induced chemokines production could represent a novel amplification loop of leukocyte recruitment, while a subset of chemokine-inducible transcripts could be involved in monocyte extravasation and tissue invasion.
Activation of Protease-Activated Receptor (PAR)-1, PAR-2, and PAR-4 Stimulates IL-6, IL-8, and Prostaglandin E2 Release from Human Respiratory Epithelial Cells
Authors: Asokananthan Nithiananthan , Graham Peter T. , Fink Joshua , Knight Darryl A. , Bakker Anthony J. , McWilliam Andrew S. , Thompson Philip J. , Stewart Geoffrey A.
Published: April 2014

Epithelia from many tissues express protease-activated receptors (PARs) that play a major role in several different physiological processes. In this study, we examined their capacity to modulate IL-6, IL-8, and PGE2 production in both the A459 and BEAS-2B cell lines and primary human bronchial epithelial cells (HBECs). All three cell types expressed PAR-1, PAR-2, PAR-3, and PAR-4, as judged by RT-PCR and immunocytochemistry. Agonist peptides corresponding to the nascent N termini of PAR-1, PAR-2, and PAR-4 induced the release of cytokines from A549, BEAS-2B, and HBECs with a rank order of potency of PAR-2 > PAR-4 > PAR-1 at 400 μM. PAR-1, PAR-2, and PAR-4 also caused the release of PGE2 from A549 and HBECs. The PAR-3 agonist peptide was inactive in all systems tested. PAR-1, PAR-2, or PAR-4, in combination, caused additive IL-6 release, but only the PAR-1 and PAR-2 combination resulted in an additive IL-8 response. PAR peptide-induced responses were accompanied by changes in intracellular calcium ion concentrations. However, Ca2+ ion shutoff was ∼2-fold slower with PAR-4 than with PAR-1 or PAR-2, suggesting differential G protein coupling. Combined, these data suggest an important role for PAR in the modulation of inflammation in the lung.
Distinct Temporal Patterns of Macrophage-Inflammatory Protein-2 and KC Chemokine Gene Expression in Surgical Injury
Authors: Endlich Brian , Armstrong David , Brodsky Jason , Novotny Michael , Hamilton Thomas A.
Published: April 2014

In the present study the regulation of CXC chemokine expression was evaluated in full-thickness abdominal wounds in mice. During the first 24 h after injury, IL-1αβ, KC, macrophage-inflammatory protein (MIP)-2, and monocyte chemoattractant protein-1 were the predominant cytokines and chemokines produced; TNF-α was not detected. Chemokine mRNA expression and protein secretion occurred in two temporal stages. The first, which reached a maximum at 6 h, was associated with high levels of IL-1α and KC and low levels of MIP-2. This stage could be reproduced by intradermal injection of IL-1α or IL-1β and was partially blocked by injection of neutralizing Ab against IL-1α but not IL-1β. In animals depleted of circulating neutrophils, chemokine expression was reduced by nearly 70% during this stage. In the second stage, which peaked at 24 h after injury, modest but significant levels of IL-1β were detected in association with low levels of KC and high levels of MIP-2. This pattern of chemokine expression could not be mimicked by injection of IL-1α or IL-1β (even with prolonged exposure), although MIP-2 expression could be partially inhibited by intradermal injection of neutralizing Ab against IL-1β. Surprisingly, neutrophil depletion before injury resulted in sustained high levels of both KC and MIP-2 expression. These observations demonstrate that these two closely related chemokines are under distinct regulatory controls in vivo that are likely to reflect the temporally ordered participation of different cell types and/or extracellular stimuli and inhibitors.
Beryllium Skin Patch Testing to Analyze T Cell Stimulation and Granulomatous Inflammation in the Lung
Authors: Fontenot Andrew P. , Maier Lisa A. , Canavera Scott J. , Hendry-Hofer Tara B. , Boguniewicz Mark , Barker Elizabeth A. , Newman Lee S. , Kotzin Brian L.
Published: April 2014

Chronic beryllium disease (CBD) is characterized by granulomatous inflammation and the accumulation of CD4+ T cells in the lung. Patch testing of CBD patients with beryllium sulfate results in granulomatous inflammation in the skin. We investigated whether the T cell clonal populations present in the lung of CBD patients would also be present in the involved skin of a positive beryllium patch test and thus mirror the granulomatous process in the lung. CBD patients with clonal TCR expansions in bronchoalveolar lavage (BAL) were selected for study. All three CBD patients studied had a positive response to beryllium sulfate application and a negative patch test to normal saline. Immunohistochemistry showed extensive infiltration with CD4+ T cells and few, if any, CD8+ T cells both at 3 days and at later times when granulomas were apparent. T cell infiltration early after skin testing appeared to be nonspecific with the TCR repertoire of infiltrating T cells being distinct from that present in BAL. At later times when granulomas were present, T cell clones in skin overlapped with those in BAL in all patients tested. Total TCR matches in skin and BAL were as high as 40% in selected Vβ T cell subsets. Studies of peripheral blood T cells before and after patch testing provided evidence for mobilization of large numbers of pathogenic beryllium-reactive T cells into the circulating pool. These studies using skin patch testing provide new insight into the dynamics of T cell influx and mobilization during granulomatous inflammation.
Long-Term Reversal of Established Autoimmunity upon Transient Blockade of the LFA-1/Intercellular Adhesion Molecule-1 Pathway
Authors: Bertry-Coussot Lydia , Lucas Bruno , Danel Claire , Halbwachs-Mecarelli Lise , Bach Jean-François , Chatenoud Lucienne , Lemarchand Patricia
Published: April 2014

Transgenic models and administration of mAbs directed against the LFA-1/intercellular adhesion molecule 1 (ICAM-1) pathway have shown that these costimulatory molecules play a key role in generating effector cells mediating inflammatory responses. In this report, durable remission of recent diabetes in nonobese diabetic (NOD) mice was induced by transient expression of an immunoadhesin gene encoding the soluble form of ICAM-1 (sICAM-1/Ig). A single i.v. injection of an adenovirus vector encoding the immunoadhesin gene led to 70% diabetes remission as opposed to 0% in mice injected with a control adenovirus vector. Despite the rapid decline of sICAM-1/Ig serum levels, diabetes remission remained stable in 50% of NOD mice for >6 mo. sICAM-1/Ig expression also led to long-term protection against diabetes in prediabetic NOD mice. sICAM-1/Ig in vitro induced an agonistic effect of T cell activation in a TCR-transgenic model, increasing T cell proliferation and IL-2 secretion. Importantly, protected mice were not immunosuppressed because they rejected skin allografts normally and developed immunity against the adenovirus vector. Rather, sICAM-1/Ig induced active tolerance, as assessed by the persistence of diabetogenic T cells in protected mice and the reversal of protection by immunosuppression with cyclophosphamide.
Oligoclonal T Cell Expansion in the Skin of Patients with Systemic Sclerosis
Authors: Sakkas Lazaros I. , Xu Bin , Artlett Carol M. , Lu Song , Jimenez Sergio A. , Platsoucas Chris D.
Published: April 2014

Fibrosis, microvascular fibroproliferative alterations, and autoantibody production are the main features of systemic sclerosis (SSc), and all of them can be explained by cytokine production by activated T cells. However, little is known about the role of T cells in the pathogenesis of SSc, and there is no information on the Ag(s) that elicits such activation. To determine whether T cells infiltrating the skin biopsies of patients with SSc are oligoclonal, β-chain TCR transcripts from T cells infiltrating the skin of five patients with SSc of recent onset were amplified by either Vβ-specific PCR or nonpalindromic adaptor PCR. The resulting PCR products were subsequently cloned and sequenced. High proportions of identical β-chain TCR transcripts ranging from 43 to 90% of those sequenced were found in five patients, strongly suggesting the presence of oligoclonal T cells in these infiltrates. A dominant T cell clone was found to be clonally expanded in skin biopsies obtained from a single patient with SSc at three different times (0, 8, and 13 mo earlier) and from three different skin regions. β-chain TCR transcripts from PBMC from normal donors (methodological control) were unique when compared with each other, typical for polyclonal populations of T cells. The finding of oligoclonal T cells infiltrating the skin of patients with SSc suggests that these T cells have undergone proliferation in situ in the skin and clonal expansion in response to as yet unidentified Ag(s). These results suggest that T cells are involved in the pathogenesis of the disease.
Cutting Edge: Chemical Dominance Does Not Relate to Immunodominance: Studies of the CD4+ T Cell Response to a Model Antigen
Authors: DiPaolo Richard J. , Unanue Emil R.
Published: April 2014

We have quantitated the major families of peptides from hen egg lysozyme (HEL) presented by MHC class II I-Ak molecules. One striking feature is that the four epitopes are presented at levels that differ by as much as 200- to 300-fold. In these studies, we describe the CD4+ T cell response to each epitope after immunization with several doses of hen egg lysozyme protein. Although fewer T cells were generated at lower doses, the surprising finding was the responses to all four peptides were maintained. The relative number of T cell clones to each of the four epitopes was influenced to a very limited degree by their levels of presentation at the lowest dose. In conclusion, under strong stimulatory conditions, there is not a direct relationship between levels of peptide presentation and the T cell responses.
Antigen Presentation Capacity and Cytokine Production by Murine Splenic Dendritic Cell Subsets upon Salmonella Encounter
Authors: Yrlid Ulf , Wick Mary Jo
Published: April 2014

Salmonella typhimurium is an intracellular bacterium that replicates in the spleen and mesenteric lymph nodes (MLN) of orally infected mice. However, little is known about the Ag presentation and cytokine production capacity of dendritic cells (DC), particularly CD8α+, CD8α−CD4−, and CD8α−CD4+ DC, from these organs in response to Salmonella. Infection of purified splenic DC with S. typhimiurium expressing green fluorescent protein (GFP) and OVA revealed that all three splenic DC subsets internalize bacteria, and splenic as well as MLN DC process Salmonella for peptide presentation. Furthermore, presentation of Salmonella Ags on MHC-I and MHC-II was evident in both CD8α+ and CD8α− splenic DC subsets. Direct ex vivo analysis of splenic DC from mice infected with GFP-expressing Salmonella showed that all three subsets harbored bacteria, and splenic DC purified from mice given Salmonella-expressing OVA presented OVA-derived peptides on MHC-I and MHC-II. Cytokine production analyzed by intracellular staining of splenic DC infected with GFP-expressing Salmonella revealed that TNF-α was produced by a large percentage of CD8α− DC, while only a minor proportion of CD8α+ DC produced this cytokine following bacterial exposure. In contrast, the greatest number of IL-12p40-producing DC were among CD8α+ DC. Experiments inhibiting bacterial uptake by cytochalasin D as well as use of a Transwell system revealed that bacterial contact, but not internalization, was required for cytokine production. Thus, DC in sites of Salmonella replication and T cell activation, spleen and MLN, respond to bacterial encounter by Ag presentation and produce cytokines in a subset-specific fashion.
Pertussis Toxin Modulates the Immune Response to Neuroantigens Injected in Incomplete Freund’s Adjuvant: Induction of Th1 Cells and Experimental Autoimmune Encephalomyelitis in the Presence of High Frequencies of Th2 Cells
Authors: Hofstetter Harald H. , Shive Carey L. , Forsthuber Thomas G.
Published: April 2014

Pertussis toxin (PT) has been widely used to facilitate the induction of experimental autoimmune encephalomyelitis (EAE) in rodents. It has been suggested that this microbial product promotes EAE by opening up the blood-brain barrier and thereby facilitates the migration of pathogenic T cells to the CNS. However, PT has other biological effects that could contribute to its activity in EAE, such as enhancing the cytokine production by T cells and induction of lymphocytosis. In this work, we investigated the effects of PT on the pathogenicity, cytokine differentiation, and clonal sizes of neuroantigen-reactive T cells in EAE in mice. Our results show that PT prevented the protection from EAE conferred by injection of PLPp139–151 in IFA and induced high frequencies of peptide-specific Th1 cells and disease. Interestingly, the mice developed EAE despite the simultaneous vigorous clonal expansion of PLPp139–151-specific Th2 cells. The data indicate that the Th2 cells in this model neither were protective against EAE nor promoted the disease. Furthermore, the results suggested that the effects of the toxin on neuroantigen-reactive T cells were promoted by the PT-induced activation of APCs in lymphoid tissues and the CNS. Together, the results suggest that microbial products, such as PT, could contribute to the initiation of autoimmune disease by modulating the interaction between the innate and adaptive immune system in the response to self Ags.
Antigen-Dependent Release of IFN-γ by Cytotoxic T Cells Up-Regulates Fas on Target Cells and Facilitates Exocytosis-Independent Specific Target Cell Lysis
Authors: Müllbacher Arno , Lobigs Mario , Hla Ron Tha , Tran Thao , Stehle Thomas , Simon Markus M.
Published: April 2014

Effector cytolytic T (Tc) lymphocytes, deficient in the exocytosis-mediated pathway of target cell lysis, induce Fas on target cells and, in turn, delayed cell death and apoptosis via the Fas ligand-Fas interaction. The induction of Fas can be blocked by anti- IFN-γ Abs. This Fas up-regulation on initially Fas-negative target cells is not mediated by TCR-MHC/peptide signaling per se, but by secreted IFN-γ from Tc cells after Ag engagement. The Fas up-regulation by Tc cells can be mimicked by treatment of target cells with rIFN-γ. Tc cells from IFN-γ knockout mice do not induce Fas expression on target cells. Tc cell-mediated Fas expression on third party, bystander, target cells does not enhance their susceptibility to lysis by these nominal effector cells. The results are discussed as to the possible relevance of the phenomenon in efficiency and regulation of the Tc cell response to infections by viruses.
Extracellular Adenine Nucleotides Inhibit the Activation of Human CD4+ T Lymphocytes
Authors: Duhant Xavier , Schandené Liliane , Bruyns Catherine , Gonzalez Nathalie Suarez , Goldman Michel , Boeynaems Jean-Marie , Communi Didier
Published: April 2014

ATP has been reported to inhibit or stimulate lymphoid cell proliferation, depending on the origin of the cells. Agents that increase cAMP, such as PGE2, inhibit human CD4+ T cell activation. We demonstrate that several ATP derivatives increase cAMP in both freshly purified and activated human peripheral blood CD4+ T cells. The rank order of potency of the various nucleotides was: adenosine 5′-O-(3-thiotriphosphate) (ATPγS) ≈ 2′- and 3′-O-(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP ≫ dATP, 2-propylthio-β,γ-dichloromethylene-d-ATP, UDP, UTP. This effect did not involve the activation of A2Rs by adenosine or the synthesis of prostaglandins. ATPγS had no effect on cytosolic calcium, whereas BzATP induced an influx of extracellular calcium. ATPγS and BzATP inhibited secretion of IL-2, IL-5, IL-10, and IFN-γ; expression of CD25; and proliferation after activation of CD4+ T cells by immobilized anti-CD3 and soluble anti-CD28 Abs, without increasing cell death. Taken together, our results suggest that extracellular adenine nucleotides inhibit CD4+ T cell activation via an increase in cAMP mediated by an unidentified P2YR, which might thus constitute a new therapeutic target in immunosuppressive treatments.
Absence of the CD1 Molecule Up-Regulates Antitumor Activity Induced by CpG Oligodeoxynucleotides in Mice
Authors: Sfondrini Lucia , Besusso Dario , Zoia Maria Teresa , Rodolfo Monica , Invernizzi Anna Maria , Taniguchi Masaru , Nakayama Toshinori , Colombo Mario Paolo , Ménard Sylvie , Balsari Andrea
Published: April 2014

The role of NKT cells on antitumor activity of CpG oligodeoxynucleotides (ODNs) was evaluated by peritumoral injections of CpG-ODNs in s.c. melanoma-bearing mice of strains differing in the number of NKT cells (athymic nude mice, recombination-activating gene−/−/transgenic Vα14/Vβ8.2 mice that generate NKT cells; Jα281−/− mice and CD1−/− mice, which both have a strongly reduced number of NKT cells; and C57BL/6 wild-type mice). Tumor growth was significantly inhibited in strains enriched or depleted of NKT cells. The two murine strains having a reduced number of NKT cells differed significantly in the CpG-dependent tumor growth inhibition: in Jα281−/− mice this inhibition was superimposable to that observed in C57BL/6 mice, while in CD1−/− mice the inhibition was dramatic. The increased tumor inhibition in CD1−/− correlated with a significantly higher ratio of IFN-γ-IL-4 production in response to CpG as compared with C57BL/6 and Jα281−/− mice. Experiments in which preparations of APCs and lymphocytes of the three strains were mixed showed that in the presence of APCs not expressing CD1, the production of CpG-ODN-induced type 1 cytokines was higher. Phenotype analysis of IFN-γ- and IL-4-producing cells revealed that the differences between CD1−/− and C57BL/6 in the production of these two cytokines were mainly due to CD3+ T lymphocytes. These data point to a regulatory role for the CD1 molecule in antitumor activity induced by danger signals, independently of Vα14 NKT cells. The identification of a CD1-dependent suppressive subpopulation(s) might have important implications for the study of tolerance in the context of cancer, autoimmunity, and transplantation.
Human Cytomegalovirus Disrupts Constitutive MHC Class II Expression
Authors: Cebulla Colleen M. , Miller Daniel M. , Zhang Yingxue , Rahill Brian M. , Zimmerman Peter , Robinson John M. , Sedmak Daniel D.
Published: April 2014

CD8+ and CD4+ T lymphocytes are important in controlling human CMV (HCMV) infection, but the virus has evolved protean mechanisms to inhibit MHC-based Ag presentation and escape T lymphocyte immunosurveillance. Herein, the interaction of HCMV with the MHC class II Ag presentation pathway was investigated in cells stably transfected with class II transactivator. Flow cytometry experiments demonstrate that HCMV infection decreases cell-surface MHC class II expression. HCMV down-regulates MHC class II surface expression without a significant effect on class II RNA or steady-state protein levels. SDS-stability and confocal microscopy experiments demonstrate normal levels of steady-state peptide-loaded class II molecules in infected cells and that class II molecules reach late endosomal and HLA-DM positive peptide-loading compartments. However, MHC class II positive vesicles are retained in an abnormal perinuclear distribution. Finally, experiments with a mutant HCMV strain demonstrate that this novel mechanism of decreased MHC class II expression is not mediated by one of the known HCMV immunomodulatory genes. These defects in MHC class II expression combined with previously identified CMV strategies for decreasing MHC class I expression enables infected cells to evade T lymphocyte immunosurveillance.
Expression and Function of the Eph A Receptors and Their Ligands Ephrins A in the Rat Thymus
Authors: Muñoz Juan J. , Alonso-C. Luis M. , Sacedón Rosa , Crompton Tessa , Vicente Angeles , Jiménez Eva , Varas Alberto , Zapata Agustín G.
Published: April 2014

Thymus development and function are dependent on the definition of different and graded microenvironments that provide the maturing T cell with the different signals that drive its maturation to a functional T lymphocyte. In these processes, cell-cell interactions, cell migration, and positioning are clues for the correct functioning of the organ. The Eph family of receptor tyrosine kinases and their ligands, the ephrins, has been implicated in all these processes by regulating cytoskeleton and adhesion functioning, but a systemic analysis of their presence and possible functional role in thymus has not yet been conducted. In this regard, the current study combines different experimental approaches for analyzing the expression of four members of the Eph A family and their ligands, ephrins A, in the embryonic and adult rat thymus. The patterns of Eph and ephrin expression in the distinct thymic regions were different but overlapping. In general, the studied Eph A were expressed on thymic epithelial cells, whereas ephrins A seem to be more restricted to thymocytes, although Eph A1 and ephrin A1 are expressed on both cell types. Furthermore, the supply of either Eph A-Fc or ephrin A-Fc fusion proteins to fetal thymus organ cultures interferes with T cell development, suggesting an important role for this family of proteins in the cell mechanisms that drive intrathymic T cell development.
Characterization of the Peptide-Binding Specificity of Mamu-B*17 and Identification of Mamu-B*17-Restricted Epitopes Derived from Simian Immunodeficiency Virus Proteins
Authors: Mothé Bianca R. , Sidney John , Dzuris John L. , Liebl Max E. , Fuenger Sarah , Watkins David I. , Sette Alessandro
Published: April 2014

The SIV-infected rhesus macaque is an excellent model to examine candidate AIDS virus vaccines. These vaccines should elicit strong CD8+ responses. Previous definition of the peptide-binding motif and optimal peptides for Mamu-A*01 has created a demand for Mamu-A*01-positive animals. We have now studied a second MHC class I molecule, Mamu-B*17, that is present in 12% of captive-bred Indian rhesus macaques. The peptide-binding specificity of the Mamu-B*17 molecule was characterized using single substitution analogs of two Mamu-B*17-binding peptides and libraries of naturally occurring sequences of viral or bacterial origin. Mamu-B*17 uses position 2 and the C terminus of its peptide ligands as dominant anchor residues. The C terminus was found to have a very narrow specificity for the bulky aromatic residue W, with other aromatic residues (F and Y) being only occasionally tolerated. Position 2 is associated with a broad chemical specificity, readily accommodating basic (H and R), bulky hydrophobic (F and M), and small aliphatic (A) residues. Using this motif, we identified 50 peptides derived from SIVmac239 that bound Mamu-B*17 with an affinity of 500 nM or better. ELISPOT and intracellular cytokine-staining assays showed that 16 of these peptides were antigenic. We have, therefore, doubled the number of MHC class I molecules for which SIV-derived binding peptides have been characterized. This allows for the quantitation of immune responses through tetramers and analysis of CD8+ function by intracellular cytokine-staining assays and ELISPOT. Furthermore, it is an important step toward the design of a multiepitope vaccine for SIV and HIV.