bluuebunny/crossref_metadata_2025 huggingface dataset:
Polyreactive antigen‐binding B cells in the peripheral circulation are IgD+ and B7−
Authors: Chen Zhi Jian , Shimizu Fumio , Wheeler Jim , Notkins Abner Louis
Published: March 2007

Polyreactive antibodies are naturally occurring antibodies, primarily of the IgM isotype, that are capable of reacting with a wide variety of different self and non‐self antigens. Previously, we reported that a B cell capable of making polyreactive antibody has Ig receptors on its surface that can bind different antigens. The present investigation was initiated to characterize these polyreactive antigen‐binding B cells further. A panel of fluorescein isothiocyanate‐labeled antigens (insulin, IgG Fc fragment or β‐galactosidase) served as probes to select polyreactive antigen‐binding B cells by cell sorting. Our experiment revealed that these polyreactive antigen‐binding B cells were mainly of the IgD isotype. They expressed high levels of CD40 and major histocompatibility complex class II molecules, but little or no B7‐1, B7‐2, or Fas. In contrast to the binding of antigens to monoreactive receptors (usually high affinity), the binding of antigens to polyreactive receptors (usually moderate or low affinity) did not up‐regulate the expression of B7‐1 or B7‐2. Antigens that bound to polyreactive receptors, however, were internalized and degraded, although not as efficiently as antigens that bound to monoreactive receptors. Despite the ability of these B7− cells to process antigens, they were not able to activate T cells in a mixed leukocyte reaction. It is concluded that polyreactive antigen‐binding B cells have properties that are consistent with the ability to induce immunological tolerance.
CD66: role in the regulation of neutrophil effector function
Authors: Stocks S. Craig , Ruchaud‐Sparagano Marie‐hélène , Kerr Michael A. , Grunert Fritz , Haslett Christopher , Dransfield Ian
Published: March 2007

Neutrophils express several heavily glycosylated carcinoembryonic antigen (CEA)‐related glycoproteins (CD66 antigens) which have been implicated in adhesion to E‐selectin and as receptors for the lectins galectin 3 and bacterial type‐1 fimbriae. The role of the CD66 antigens in neutrophil effector function was examined using non‐cross‐reacting and cross‐reacting domain‐mapped CD66 monoclonal antibody (mAb), which recognize epitopes on biliary glycoprotein (BGP; CD66a), CEA gene family member 6 (CGM6; CD66b), nonspecific cross‐reacting antigen 90 (NCA90; CD66c) or CGM1 (CD66d). We show that BGP‐specific mAb which recognize an AB‐domain epitope strongly augment adhesion to fibrinogen by an Fc receptor‐ and β2 integrin‐dependent mechanism. Co‐ligation of BGP with the glycophosphatidylinositol (GPI)‐anchored CGM6 and NCA90 also caused increased β2 integrin‐mediated adhesion, receptor clustering and priming of formyl‐Met‐Leu‐Phe (fMLP)‐induced oxidant production by neutrophils, but only a small change in expression of L‐selectin and CR3 compared to the chemotactic peptide fMLP. Ligation of CGM6 or NCA90 alone did not cause activation of the neutrophil in any of the assays used and did not cause priming of fMLP‐induced oxidant production even when a secondary cross‐linking reagent was used. We propose that specific cross‐linking of neutrophil BGP with CGM6 and NCA90 contributes significantly to the regulation of neutrophil function during neutrophil recruitment.
Reduced incidence and severity of collagen‐induced arthritis in interleukin‐12‐deficient mice
Authors: McIntyre Kim W. , Shuster David J. , Gillooly Kathleen M. , Warrier Rajeev R. , Connaughton Suzanne E. , Hall Leroy B. , Arp Lawrence H. , Gately Maurice K. , Magram Jeanne
Published: March 2007

Collagen‐induced arthritis (CIA) is an animal model for rheumatoid arthritis. The disease is elicited by immunization of genetically susceptible DBA/1 mice with type II collagen, resulting in a debilitating arthritis characterized by inflammation and involvement of multiple joints. We investigated the role of endogenous interleukin (IL)‐12 in the pathogenesis of this disease by undertaking an analysis of IL‐12‐deficient mice on the DBA/1 genetic background after immunization with type II collagen. Both the incidence and severity of disease were significantly reduced in mice unable to produce biologically active IL‐12. Concomitant decreases were observed in serum levels of pathogenic, collagen‐specific IgG2a antibodies and collagen‐induced secretion of interferon‐γ by immune splenocytes in vitro, consistent with an impaired T helper‐1 response. There were, however, a few animals which developed severe disease in a single paw in spite of this highly diminished Th1 response. Taken together, these results demonstrate an important role for IL‐12 in the pathogenesis of CIA, although it is not absolutely required for disease development.
Heat shock increases antigenic peptide generation but decreases antigen presentation
Authors: Pépin Elsa , Villiers Christian L. , Gabert Françise M. , Serra Vincent A. , Marche Patrice N. , Colomb Maurice G.
Published: March 2007

The heat shock response is a universal and highly conserved cellular response to stress. We describe here the effect of elevated temperature on the capacity of B cells to present antigen. Heat shock markedly affects the ability of these cells to process and present tetanus toxin to class II‐restricted T cell clones. Inhibition of antigen presentation is due neither to a modification of antigen capture nor to a variation of major histocompatibility complex (MHC) class II molecule synthesis and cell surface expression. Stressed and nonstressed B cells are able to present peptides loaded at the cell surface with the same efficiency. Nevertheless, heat shock leads to an increase of antigen peptide generation in subcellular compartments; an enhancement of cathepsin B activity is also observed. These data suggest that such a stress induces a failure in the intracellular peptide loading onto MHC class II molecules.
Early quantitative and functional deficiency of NK1+‐like thymocytes in the NOD mouse
Authors: Gombert Jean‐Marc , Herbelin André , Tancrède‐Bohin Emmanuelle , Dy Michel , Carnaud Claude , Bach Jean‐François
Published: March 2007

An immunoregulatory role has recently been attributed to the discrete subset of major histocompatibility complex class I‐restricted NK1+ mature heat‐stable antigen− (HSA−) thymocytes expressing an unusual Vβ8‐biased T cell receptor repertoire. NK1+ T cells are the main interleukin (IL)‐4 producers upon priming. We have studied the size and the function of this subset in the nonobese diabetic (NOD) mouse, a model of spontaneous T cell‐mediated autoimmune insulin‐dependent diabetes. This study was complicated by the absence in this strain of the NK1.1 allele, the only one for which an antibody is available. To circumvent this difficulty, the cells, hereafter designated the NK1+‐like T subset, were characterized by the use of monoclonal antibodies which showed the Vβ8 bias in the CD44+ Ly‐49+ MEL‐14− 3G11− thymocyte subset of non‐autoimmune strains and of its absence in class I‐deficient (β2‐microglobulin−/−) mice. A clear deficit in the number of NK1+‐like cells was evidenced at 3 weeks of age in NOD mice. It was still present at 8 weeks of age in the double‐negative CD4−CD8− population. The functional anomaly was even more striking: NOD mouse NK1+‐like thymocytes virtually lacked the ability to produce IL‐4 at 3 weeks and still showed a very reduced capacity at 8 weeks. NK1+ T cell deficiency was also suggested in the periphery by the reduction of Ly‐49A+ cells in the spleen of 3‐ and 8‐week‐old NOD mice and the absence of short‐term production of IL‐4 in vitro by NOD mouse spleen cells 90 min after the administration of anti‐CD3 antibody, a response attributed to NK1+ T cells. Taken together, these data demonstrate a very early defect in NK1+‐like T cells which could be involved in the genesis of autoimmunity in NOD mice through a deficiency in Th2 cell function.
Defective antigen processing correlates with a low level of intracellular glutathione
Authors: Short Suzanna , Merkel Brian J. , Caffrey Rebecca , McCoy Kathleen L.
Published: March 2007

Previously, we reported that Chinese hamster ovary (CHO) cells transfected with murine mouse major histocompatibility complex class II genes, exhibit a unique antigen (Ag) processing defect whereby these cells are impaired in processing only Ag with disulfide bonds. Here, we examined various aspects of the intracellular reducing environment in the CHO cells to understand the underlying mechanism causing the defect. A cell hybrid generated by the fusion of CHO cells and L cell fibroblasts was used for comparison due to their competency in processing Ag. The transport pathway of cysteine within the CHO cells appeared normal. However, these cells had a significantly lower level of glutathione, a major physiological reducing thiol, compared to the cell hybrid. Treatment of the CHO cells with N‐acetyl‐L‐cysteine did not augment their glutathione content nor their ability to process Ag. When the cell hybrid was treated with L‐buthionine‐(S,R)‐sulfoximine (BSO), which significantly decreased their glutathione level, the hybrid poorly processed hen egg lysozyme (HEL) and ovalbumin, which have disulfide bonds. In contrast, BSO treatment did not affect the capacity of the hybrid to process pigeon cytochrome c and carboxymethylated HEL, which lack disulfide bonds. Therefore, low intracellular glutathione levels in antigen‐presenting cells correlated with defective processing of Ag with disulfide bonds, indicating that this thiol may be a critical factor in regulating productive Ag processing.
CD40‐mediated stimulation of B1 and B2 cells: implication in autoantibody production in murine lupus
Authors: Kaneko Yoriaki , Hirose Sachiko , Abe Masaaki , Yagita Hideo , Okumura Ko , Shirai Toshikazu
Published: March 2007

B1 cells usually show preferential responses to T cell‐independent antigens. To ask whether B1 cells could respond to CD40‐mediated stimulation for proliferation and differentiation, and whether CD40‐mediated signals are involved in the production of autoantibodies by B1 cells, we compared responses to our newly established agonistic anti‐mouse CD40 monoclonal antibody (mAb) between B1 and B2 cells from autoimmune‐prone (NZB X NZW) F1 mice. Stimulation with this mAb induced a similar level of proliferative responses of both B1 and B2 cells, as well as an increase in expression of cell surface molecules I‐A, CD54, CD23, CD80, and CD86. While co‐stimulation with interleukin (IL)‐4 markedly augmented proliferative as well as IgG1 and IgE antibody responses of both B1 and B2 cells, co‐stimulation with IL‐5 augmented proliferative and IgM antibody responses of only B1 cells. Splenic B1, but not B2 cells from young (NZB X NZW) F1 mice spontaneously produced substantial amounts of IgM including IgM anti‐DNA antibodies, and the levels increased in case of stimulation with anti‐CD40 mAb alone, or to a greater extent with the mAb plus IL‐4 and IL‐5. Collectively, these results indicate that splenic B1 cells from autoimmune (NZB X NZW) F1 mice have a comparable responsiveness to the CD40‐mediated stimulation to that of B2 cells, which would be a potent regulatory mechanism involved in the spontaneous production of autoantibodies by B1 cells.
Effect of CD80 and CD86 blockade and anti‐interleukin‐12 treatment on mouse acute graftversus‐host disease
Authors: Saito Kiyoshi , Yagita Hideo , Hashimoto Hiroshi , Okumura Ko , Azuma Miyuki
Published: March 2007

We investigated the efficacy of a combination of anti‐CD80 and CD86 (CD80 + 86) monoclonal antibodies (mAb), anti‐interleukin (IL)‐12 mAb, or both, for prophylaxis in a mouse acute graft‐versus‐host‐disease (GVHD) model. The treatment with a combination of anti‐CD80 + 86 mAb efficiently reduced the lethality of GVHD, whereas mAb against either CD80 or CD86 alone had an effect. A delay in lymphocyte reconstitution and GVHD‐associated histological changes in organs was observed at 30 days post‐bone marrow transplantation (BMT) even in the anti‐CD80 + 86 mAb‐treated mice, although these manifestations were resolved by 100 days. In vitro, host alloantigen‐specific T cell proliferative responses and generation of CTL were significantly reduced by anti‐CD80 + 86 treatment. Furthermore, anti‐CD80 + 86 mAb preferentially inhibited the production of interferon (IFN)‐γ, but not IL‐4 and IL‐10, when cultures were assayed at 21 days. Although the anti‐IL‐12 mAb treatment alone inhibited the generation of cytotoxic T lymphocytes and IFN‐γ production in vitro, administration of anti‐IL‐12 mAb in vivo reversed the beneficial effects of anti‐CD80 + 86 treatment on host survival post‐BMT. The adverse effect of anti‐IL‐12 treatment seems to result from impairment of natural immunity and hematopoiesis, rather than as a consequence of an incomplete blockade of T helper (Th)1 responses. Our results suggest that the prevention of GVHD‐induced death results from the efficient blockade of Th1 cell activation by the anti‐CD80 + 86 treatment. However, further treatment is required for a complete prevention of GVHD, which seems to be partly mediated by Th2 cells.
RAG1, RAG2 and pre‐T cell receptor α chain expression by adult human hepatic T cells: evidence for extrathymic T cell maturation
Authors: Collins Clive , Norris Suzanne , McEntee Gerry , Traynor Oscar , Bruno Ludovica , von Boehmer Harald , Hegarty John , O'Farrelly Cliona
Published: March 2007

Flow cytometric analysis of cell suspensions obtained from normal adult liver tissue at the time of transplantation revealed significant populations of T lymphocytes. These were examined for molecular evidence of local T cell maturation using reverse transcription‐polymerase chain reaction to detect expression of recombination activation gene 1 (RAG1), RAG2 and pre‐T cell receptor α chain (pTα), which occurs only in early thymocyte development. Four specimens of whole liver were positive for RAG1 and RAG2 expression, whereas peripheral blood mononuclear cells from the same individuals were negative. To localize RAG expression, immature (CD2+CD7+) and mature (CD45R0+) T cell subpopulations were isolated by magnetic separation from hepatic and peripheral blood mononuclear cell preparations. We detected the expression of RAG1, RAG2 and pre‐TCRα in five specimens of hepatic CD2+CD7+ but not in CD45RO+ hepatic lymphocytes. Four out of six specimens of CD2+CD7+ cells from the peripheral blood were negative for RAG1 and RAG2 while all six specimens were positive for pTα expression. These results suggest that pre‐T cells are trafficking from the bone marrow or the thymus to other tissues to continue differentiation and selection in the context of an appropriate cellular and molecular environment. The presence of immature populations of T cells in the adult liver and high levels of RAG expression suggests that the adult liver provides such an environment for extrathymic T cell maturation. These findings may have important implications for tolerance induction after liver transplantation and offer help in understanding the etiology of autoimmune liver disease.
Intracellular co‐localization of Trypanosoma cruzi and inducible nitric oxide synthase (iNOS): evidence for dual pathway of iNOS induction
Authors: Rottenberg Martín E. , Castaños‐Velez Esmeralda , De Mesquita Roberto , Laguardia Oscar Goñi , Biberfeld Peter , Örn Anders
Published: March 2007

Evidence is presented from studies in vitro and in vivo for a dual pathway of inducible nitric oxide synthase (iNOS) induction during Trypanosoma cruzi infection, one of which is interferon (IFN)‐γ dependent and the other not. In vitro, the IFN‐γ‐dependent iNOS induction decreases parasite multiplication, and is in vivo associated with protection. iNOS induced by this pathway mediated a high NO output and showed a diffuse, cytoplasmic immunostaining in IFN‐γ‐activated macrophages in vitro as well as in cell infiltrates or infected tissues. Surprisingly, in such tissues, iNOS co‐localized with parasite nests, and by immunoelectromicroscopy, iNOS was demonstrated on the parasite surface. iNOS co‐localization with parasites was also seen in tissues from T. cruzi‐infected IFN‐γ receptor (R) knockout mice suggesting an IFN‐γ‐independent pathway of induction. However, no cytoplasmic iNOS was seen in inflammatory infiltrates of these tissues. IFN‐γR−/− mice displayed a dramatically enhanced susceptibility to infection with T. cruzi, diminished accumulation of iNOS mRNA in skeletal muscle and spleen cells, and reduced release of NO and per‐oxynitrite. Expression of iNOS around intracellular parasites was also observed after infection of peritoneal macrophages or L‐929 fibroblasts in vitro in the absence of other exogenous stimuli. A time‐dependent NO release and enhanced accumulation of iNOS mRNA also was observed in infected peritoneal cells and fibroblasts. Cultured T. cruzi amastigotes, trypomastigotes, and epimastigotes were not labeled by the anti‐iNOS antibodies and contained no iNOS mRNA, indicating that the iNOS detected actually originated from the mammalian cell. A pathogenic effect of low NO levels is suggested by the arresting effect of NOS inhibitors and the enhancing consequences of low concentrations of NO donors on intracellular parasite multiplication.
Protective major histocompatibility complex genes and the role of interleukin‐4 in collagen‐induced arthritis
Authors: Hesse Matthias , Bayrak Şinasi , Mitchison Avrion
Published: March 2007

To investigate the role of interleukin (IL)‐4 during the triggering of collagen‐induced arthritis, we examined the effects of the I‐Ab and I‐E protective/suppressive genes and passively administered anti‐IL‐4 monoclonal antibody. In contrast to the action of I‐E expression on its own, which has mainly a suppressive effect post‐triggering, the combination of I‐Ab and I‐E had a marked protective effect. Assuming, on the basis of previous experience with the I‐Ab allele, that it might act through suppressing early IL‐4 production, we treated mice with the 11B11 IL‐4‐neutralizing antibody around the time of initial immunization with collagen. Treatment over a period extending to 6 days post‐immunization exacerbated the arthritis, but when curtailed to 2 days post‐immunization (and tested in pristane‐primed animals), the disease was reduced. We conclude that IL‐4 plays an essential role in triggering the disease.
Germinal center B cells rescued from apoptosis by CD40 ligation or attachment to follicular dendritic cells, but not by engagement of surface immunoglobulin or adhesion receptors, become resistant to CD95‐induced apoptosis
Authors: Koopman Gerrit , Keehnen Robert M. J. , Lindhout Ernst , Zhou David F. H. , De Groot Cornelis , Pals Steven T.
Published: March 2007

Germinal centers (GC) constitute a specialized microenvironment essential for the formation of memory B cells, B cell affinity maturation and isotype switching. Within the GC, the B cells closely interact with follicular dendritic cells (FDC) and T cells, which both provide stimuli to the B cells that prevent their entry into apoptosis and promote their differentiation into memory cells or plasma cells. Cross‐linking of B cell immunoglobulin (Ig) receptors by antigen, stimulation of the integrin adhesion molecules LFA‐1 and VLA‐4 on the B cell through interaction with their counter receptors ICAM‐1 and VCAM‐1 on the FDC and cross‐linking of CD40 on the B cells through interaction with the CD40 ligand (CD40L) on T cells have been shown to prevent entry into apoptosis of GC B cells. Triggering of CD95, on the other hand, has been shown to induce apoptosis. We therefore investigated the interaction between adhesion‐mediated signals, Ig, CD40, and CD95. The spontaneous apoptosis of GC B cells was not further increased by adding anti‐CD95. However. CD95 stimulation did result in apoptosis of GC B cells in the presence of anti‐Ig or adhesion‐mediated rescue signals, which indicates that CD95 expressed on GC B cells is functionally active. In contrast, anti‐CD95 was unable to induce apoptosis in cells rescued via CD40 stimulation, suggesting an important role for CD40L expressed on GC T cells in apoptosis regulation. We also studied apoptosis of B cells adhering to FDC, and found that B cells that interact with FDC were also rescued from CD95‐induced apoptosis. A human CD40.Fcu fusion protein that blocks CD40 ligation failed to inhibit this effect. Our studies therefore indicate that neither CD40, Ig receptors, nor adhesion receptors mediate rescue from apoptosis by FDC.
Functional relevance during lymphocyte migration and cellular localization of activated β1 integrins
Authors: Gómez Manuel , Luque Alfonso , Del Pozo Miguel A. , Hogg Nancy , Sánchez‐Madrid Francisco , Cabañas Carlos
Published: March 2007

The state of integrin activation can be assessed by monoclonal antibodies (mAb) that selectively recognize integrins in their active form. We demonstrate herein that the expression of the epitope recognized by mAb HUTS‐21 is induced on T lymphoblasts upon binding of soluble vascular cell adhesion molecule (VCAM)‐1 and an 80‐kDa tryptic fragment of fibronectin (FN80) to the β1 integrins very late activation antigen (VLA)‐4 and VLA‐5, and that this effect is dependent on ligand concentration and is specific for β1 integrins. On T lymphoblasts adhering to immobilized fibronectin, the HUTS‐21 epitope localized exclusively to sites of integrin binding to fibronectin. These results indicate that mAb HUTS‐21 recognizes a ligand‐induced binding site (LIBS) on the common β1 subunit of VLA proteins. Engagement of β1 integrins through this LIBS epitope inhibited T lym‐phoblast movement on fibronectin, as determined by quantitative time‐lapse video microscopy studies. Furthermore, the HUTS‐21 mAb also prevented T lymphoblast‐directed migration through gradients of substratum‐immobilized β1 integrin ligands such as fibronectin or VCAM‐1, whereas it did not affect migration on intercellular adhesion molecule (ICAM)‐1. This anti‐LIBS mAb stimulated cell adhesion through postreceptor events, without affecting receptor affinity for ligand, and appears to interfere with cell migration by a mechanism distinct from that of other anti‐β1 activating antibodies.
Expression of pro‐inflammatory cytokines by TCRαβ+ T and TCRγδ+ T cells in an experimental model of colitis
Authors: Simpson Stephen J. , Holländer Georg A. , Mizoguchi Emiko , Allen Deborah , Bhan Atul K. , Wang Baoping , Terhorst Cox
Published: March 2007

An inflammatory bowel disease (IBD) comparable to human ulcerative colitis is induced upon transfer of T cell‐depleted wild‐type (F1) bone marrow into syngeneic T cell‐deficient (tgε26) mice (F1 → tgε26). Previously we have shown that activated CD4+ T cells predominate in transplanted tgε26 mice, and adoptive transfer experiments verified the potential of these cells to cause disease in immunodeficient recipient mice. Using flow cytometry for the detection of intracellular cytokine expression, we demonstrate in the present study that large numbers of CD4+ and CD8+ TCRαβ+ T cells from the intraepithelial region and lamina propria of the colon of diseased, but not from disease‐free mice, produced interferon‐γ (IFN‐γ) and tumor necrosis factor‐α (TNF‐α). Large numbers of T cells from peripheral lymphoid tissues of these animals also expressed IFN‐α and TNF‐α, but few expressed interleukin‐4, demonstrating g strong bias towards Th1‐type T cell responses in these animals. TCRγδ+ T cells, typically minor constituents of the inflammatory infiltrate of the colon in F1 → tgε26 mice, also expressed IFN‐γ at a high frequency upon CD3 stimulation. In light of these findings we examined the potential involvement of TCRγδ+ T cells by testing their ability to induce colitis in tgε26 mice. We report here that tgε26 mice transplanted with T cell‐depleted bone marrow from TCRαnull and TCRβnull animals developed IBD. Furthermore, disease in these mice correlated with the development of peripheral and colonic TCRαδ+ T cells capable of IFN‐γ production. These results suggest that IFN‐γ may be a common mediator of IBD utilized by pathogenic T cells of distinct phenotype.
The influence of granulocyte/macrophage colony‐stimulating factor on dendritic cell levels in mouse lymphoid organs
Authors: Vremec David , Lieschke Graham J. , Dunn Ashley R. , Robb Lorraine , Metcalf Donald , Shortman Ken
Published: March 2007

To ascertain whether the development of dendritic cells (DC) in mouse lymphoid organs is dependent on granulocyte/macrophage colony‐stimulating factor (GM‐CSF), we determined the number of DC in the thymus, spleen and lymph nodes of normal mice, of mice with the genes coding for GM‐CSF or its receptor inactivated, and of transgenic mice with excessive levels of GM‐CSF. DC were extracted from the tissues and enriched prior to flow cytometric analysis. The total DC level and the incidence of DC expressing lymphoid‐related markers (CD8hiCD11blo) and myeloid‐related markers (CD8loCD11bhi) were monitored. Both in GM‐CSF null mice, and GM‐CSF receptor null mice, DC of all surface phenotypes were present in all lymphoid organs; only small decreases in DC levels were recorded, except for the lymph nodes of GM‐CSF receptor null mice which showed a more pronounced (threefold) decrease in DC numbers. Since the GM‐CSF receptor null mice lacked the β chain common to the GM‐CSF, interleukin (IL)‐3 and IL‐5 receptors, the development of DC in the absence of GM‐CSF was not due to common β chain mediated developmental signals elicited by IL‐3 or IL‐5. In GM‐CSF transgenic mice, there was only a 50 % increase in DC numbers in thymus and spleen, paralleling an increase in overall cellularity, but a more pronounced (threefold) increase in DC numbers in lymph nodes. There was no evidence that GM‐CSF had a selective effect on any particular DC subpopulation defined by CD8 or CD11b expression. We conclude that the development of most lymphoid tissue DC can proceed in the absence of GM‐CSF, although this cytokine can produce some elevation of DC levels. It is not clear whether the enhancing effect of GM‐CSF is direct or an indirect effect mediated by other cytokines.
Co‐receptor‐independent signal transduction in a mismatched CD8+ major histocompatibility complex class II‐specific allogeneic cytotoxic T lymphocyte
Authors: Eshima Koji , Tachibana Makoto , Suzuki Harumi , Yamazaki Sunao , Shinohara Nobukata
Published: March 2007

The contribution of co‐receptors in signal transduction upon T cell receptor (TCR)‐mediated recognition of major histocompatibility complex (MHC) class II antigen by mature T lymphocytes expressing TCR derived from the apparently co‐receptor‐independent, I‐Ak‐specific allogeneic CD8+ CTL clone QM11 has been examined. Mature double‐negative, CD8+ and CD4+ bulk T cell lines and clones expressing TCRQM11 were developed from TCRQM11 transgenic mice. All these T cells, irrespective of co‐receptor expression, showed specific lytic activity on cells expressing I‐Ak. Furthermore, co‐receptorless mutants were obtained from a CD4+ and CD8+ clone. The responses of these co‐receptorless mutants upon specific recognition of the alloantigen, as judged by cytolytic activity, granule exocytosis, lymphokine production, proliferation, and tyrosine phos‐phorylation of the ξ chain, were comparable to those of the original clones. Thus, the results proved the co‐receptor independence of the recognition of I‐Ak by TCRQM11 and further indicated there is no indispensable unique signal transduced by co‐receptors. However, when the amount of the available antigen was limited by anti‐I‐Ak antibody, the CD4+ T cell clone showed a remarkable resistance to the inhibition whereas the mismatched CD8+ clone was readily inhibitable. The anti‐I‐Ak‐resistant component of the CD4+ clone showed dependency on the CD4 molecule. Taken collectively, the results indicate that the role played by a co‐receptor molecule in mature T cells is purely quantitative amplification of the signal through the formation of a TCR/MHC/co‐receptor ternary complex, and also indicate that the role of co‐receptor molecules as TCR‐independent adhesion molecules is at best minimal.
Vα domain modulates the multiple topologies of mouse T cell receptor Vβ20/staphylococcal enterotoxins A and E complexes
Authors: De Alba Yolanda Bravo , Marche Patrice N. , Cazenave Pierre‐André , Cloutier Isabelle , Sekaly Rafick‐Pierre , Thibodeau Jacques
Published: March 2007

The superantigens staphylococcal enterotoxin A and E (SEA and SEE) both contact major histocompatibility complex (MHC) class II molecules on two sites located on the α and β chains. We have investigated the role of the T cell receptor (TCR) α chain in the modulation of the various topologies of TCR/SEA (or SEE)/class II complexes. For this purpose, we have used three mouse Vβ20 T cell lines expressing different Vα domains and two T cell hybridomas expressing mouse Vβ1 or Vβ11 segments. The response of these T cells to SEA and SEE was studied in the context of presentation by wild‐type human MHC class II molecules; or by mutants on MHC, in each of the two superantigen binding sites (position α39K and β81H) to which the superantigens can still bind but with an altered conformation. Although Vβ20 T cell lines are efficiently stimulated using SEA and SEE presented by wild‐type HLA‐DR1 molecules, our results show that the nature of the TCR Vα domain can affect differently the recognition of the toxins bound to mutant class II molecules. This suggests that various functional topologies exist for both SEA and SEE/class II complexes and that the T cell response to each of these complexes can be modulated by the Vα domain of the TCR. Interestingly, the recognition of SEA and SEE is achieved in different fashions by a given Vβ20 T cell line.
A γδ T cell specific surface receptor (WC1) signaling G0/G1 cell cycle arrest
Authors: Takamatsu Haru‐Hisa , Kirkham Paul A. , Parkhouse R. Michael E.
Published: March 2007

Three monoclonal antibodies (mAb; SC‐6, SC‐12, and SC‐29) reactive with the γδ T cell‐restricted antigen WC1 were obtained immunizing mice with an ovine interleukin (IL)‐2‐dependent γδ T cell line. These mAb strongly inhibited DNA synthesis in IL‐2‐dependent γδ T cell lines with cell cycle arrest in G0/G1 phase, but did not induce apoptosis. The mAb‐induced growth arrest was reversible, either by removing the mAb or by co‐culture with mitogen or anti‐CD3 in the presence of IL‐2. In contrast, addition of phorbol ester, ionomycin and IL‐2 had no effect on the mAb‐induced growth arrest. The observations define a biologically important role for the cell surface molecule WC1 in the regulation of γδ T cell proliferation and also provide a suitable system to study the relevant signal transduction events.
Thymic stromal organization is regulated by the specificity of T cell receptor/major histocompatibility complex interactions
Authors: Brabb Thea , Huseby Eric S. , Morgan Todd M. , Sant'Angelo Derek B. , Kirchner Jacqueline , Farr Andrew G. , Goverman Joan
Published: March 2007

The thymic architecture is normally compartmentalized into a central medulla surrounded by a peripheral cortical region. We investigated how compartmentalization of the thymic stroma is regulated using T cell receptor (TCR)‐transgenic mouse models. Our studies show that the signals generated by TCR/peptide/major histocompatibility complex interactions regulate thymic stromal cell compartmentalization. In TCR‐transgenic mice, normal stromal cell compartmentalization occurs when the transgenic TCR is expressed on a background that does not result in skewing toward either positive or negative selection. In models representing strong positive selection, the thymic stromal elements do not fully organize into a central medulla. Instead, small medullary foci are dispersed throughout the thymus with some regions residing directly under the capsule. The highest degree of disorganization in medullary epithelial regions is observed in TCR‐transgenic mice that exhibit negative selection. Although the medullary foci lack central organization, the expression in these regions of CD80, CD86 and CD40, as well as the clustering of dendritic cells, is similar to that observed in medullae of wild‐type mice. Thus, the organization of the medulla appears to occur in two stages: (1) small medullary epithelial regions that are dispersed in fetal thymi expand and associate with antigen‐presenting cells, and (2) the expanded medullary foci organize into a central medullary compartment. Our data suggest a model in which this second stage of stromal cell organization is increasingly inhibited as the normal balance of TCR‐mediated signals is skewed by higher‐avidity interactions between thymocytes and antigen‐presenting cells.
Human Th1 cells preferentially induce interleukin (IL)‐1β while Th2 cells induce IL‐1 receptor antagonist production upon cell/cell contact with monocytes
Authors: Chizzolini Carlo , Chicheportiche Rachel , Burger Danielle , Dayer Jean‐Michel
Published: March 2007

The role of human T cells in the induction and regulation, upon cell/cell contact, of inflammatory responses by monocytic cells was investigated. The production of interleukin (IL)‐1β and IL‐1 receptor antagonist (IL‐1Ra) by the monocytic THP‐1 cell line was measured upon contact with either Th1 or Th2 cell clones. CD4+ T cell clones specific for purified protein derivative of Mycobacterium tuberculosis, predominantly Th1 [high interferon (IFN)‐γ and low IL‐4 producers], or tetanus toxoid, predominantly Th2 (low IFN‐γ and high IL‐4 producers), were generated. Cell membranes from antigen‐stimulated, but not from resting T cell clones induced dose‐dependent cytokine production by THP‐1 cells. Th1 clones induced higher levels of IL‐1β production (484–806 pg/ml) than did Th2 clones (21–114 pg/ml). In contrast, Th1 clones induced lower levels of IL‐1Ra (0.9–7.8 ng/ml) than did Th2 clones (7.0–49.6 ng/ml). Similar results were obtained when T cell clones were activated by cross‐linked CD3 and CD28. IL‐1β production by THP‐1 cells correlated with IFN‐γ production by T cell clones but was unaffected by IFN‐γ neutralization. IL‐1Ra production by THP‐1 cells correlated with IL‐4 production by T cells and was partially inhibited by IL‐4 neutralization. These data indicate that activated Th1 and Th2 cells express different molecules on the cell surface able to induce distinct pro‐inflammatory (IL‐1β) or anti‐inflammatory (IL‐1Ra) responses in monocytes. This differential induction of molecules with opposite effects on inflammation stresses the functional heterogeneity in CD4+ T cells.
Bacille Calmette Guérin and interleukin‐12 down‐modulate interleukin‐4‐producing CD4+ NK1+ T lymphocytes
Authors: Emoto Masashi , Emoto Yoshiko , Kaufmann Stefan H. E.
Published: March 2007

Early production of interleukin‐12 (IL‐12) by macrophages and of IL‐4 from CD4+ NK1+ T cells influence development of the acquired immune response against infectious agents, namely differentiation of interferon‐γ‐secreting T helper 1 (Th1) cells against intracellular pathogens and of IL‐4‐producing Th2 cells against helminths. Evidence has been presented for transient convertibility of Th1 and Th2 cells in the presence of the polarizing cytokines IL‐4 or IL‐12, respectively. Moreover, it is likely that IL‐4 dominates over IL‐12, suggesting that Th2 cell development is preferred in the presence of both cytokines. Mycobacterium bovis Bacille Calmette Guérin (BCG) and IL‐12 are potent inducers of Th1 responses. Here we show that BCG and IL‐12 down‐modulate IL‐4‐producing CD4+ NK1+ TCRα/βintermediate liver lymphocytes. Our data provide further insights into the mechanisms by which BCG and IL‐12 may promote unrestricted development of Th1 responses in vivo: BCG and IL‐12 not only provide the positive stimuli for Th1 cell differentiation, but also interfere with antagonizing signals.
γδ cells involved in contact sensitivity preferentially rearrange the Vγ3 region and require interleukin‐7
Authors: Dieli Francesco , Asherson Geoffrey L. , Sireci Guido , Dominici Roberto , Gervasi Francesco , Vendetti Silvia , Colizzi Vittorio , Salerno Alfredo
Published: March 2007

Ptak and Askenase showed that both αβ and γδ cells are required for transfer of contact sensitivity (CS). This study confirms that day 4 immune cells depleted of γδ cells fail to transfer CS to trinitrochlorobenzene (TNP‐Cl) systemically and demonstrates that administration of anti‐γδ monoclonal antibodies (mAb) in vivo abolishes the CS reaction. Moreover, γδ cells accumulate at the antigen challenge site: these cells have the unusual phenotype CD8α+, CD8β‐, IL‐4 R+ which we suggest is due to their state of activation. Following immunization with contact sensitizer on the skin, the absolute number of γδ cells increases in the regional lymph nodes with a peak at 4 days. Of the γδ cells, 80%, both in the lymph nodes of TNP‐Cl‐immune mice and accumulating at the antigen challenge site are Vγ3+. The γδ cells expressing Vγ3, which is characteristic of dendritic epithelial T cells (DETC), obtained 4 days after sensitization, proliferate in response to interleukin (IL)‐7, but only poorly to IL‐2 and IL‐4. They also respond to concanavalin A and immobilized anti‐γδ mAb, but not to haptens or heat‐shocked syngeneic spleen cells. Furthermore, injection of mice with mAb to IL‐7 inhibits accumulation of Vγ3+ cells both in the lymph nodes after skin sensitization and at the antigen‐challenge site. Altogether, these results strongly support the view that DETC are related to, or the original source of, the γδ cells found in the lymph node after skin sensitization and at the site of challenge, and that IL‐7 is implicated in these phenomena.
Overexpression of mitogen‐activated protein kinase kinase kinase reversed cAMP inhibiton of NF‐χB in T cells
Authors: Ho Huang‐Yao , Lee Hsiu‐Hsiang , Lai Ming‐Zong
Published: March 2007

cAMP inhibits T cell activation by acting as an antagonist for selective kinases and transcriptional factors. We have recently demonstrated that cAMP inhibited c‐Jun N‐terminal kinase (JNK) but left the mitogen‐activated protein (MAP) kinase cascade almost unaffected in T lymphocytes. In accordance with recent reports, we also observed a selective suppression of nuclear factor NF‐χB activation by cAMP. The possible link between the JNK cascade and NF‐χB activation was demonstrated by the fact that the active form of MAP kinase kinase kinase (ΔMEKK), a constitutive activator of JNK, induced NF‐χB but not AP‐1, Oct, and NF‐AT in T cells. In contrast, the induction of MAP kinase kinase (MEK)‐MAP kinase did not stimulate NF‐χB activity. The specific activation of NF‐χB by a single MEKK‐JNK cascade was thus unusual, given that the activation of other transcriptional elements in T cells requires at least two signal pathways. This was further confirmed by the fact that cAMP inhibition of NF‐χB activation was reversed by overexpression of ΔMEKK.
The propensity to apoptosis of centrocytes and centroblasts correlates with elevated levels of intracellular myc protein
Authors: Cutrona Giovanna , Dono Mariella , Pastorino Sandra , Ulivi Massimo , Burgio Vito L. , Zupo Simona , Roncella Silvio , Ferrarini Manlio
Published: March 2007

In this study, we investigated the c‐myc expression by tonsillar germinal center (GC) B cells using reverse transcriptase‐polymerase chain reaction, flow cytometry, Western blot and in situ immunohistochemical methods. The results obtained demonstrate elevated levels of c‐myc mRNA and of Myc protein in GC B cells compared to those of the other resting or activated tonsillar B cells. Separation of GC B cells into centroblasts and centrocytes revealed that, while differing in their cell cycle status, surface marker expression and morphology, the two cell types had the same propensity to apoptosis and elevated Myc protein expression, thus reinforcing the notion of a close correlation between these two events. Based upon these observations and other considerations it is proposed that elevation of Myc proteins confers to GC B cells a particular propensity to apoptosis, while the subsequent decision between progression into the cell cycle or programmed cell death is dictated by other signals that are delivered in the GC and perhaps operate at the level of other proto‐oncogenes.
CD73 mediates lymphocyte binding to vascular endothelium in inflamed human skin
Authors: Arvilommi Anna‐Maija , Salmi Marko , Airas Laura , Kalimo Kirsti , Jalkanen Sirpa
Published: March 2007

Inflammatory diseases of the skin are characterized by abundant lymphocytic infiltrates at the site of inflammation, which are critical for the perpetuation of chronic disease. Lymphocytes gain entry to the site of inflammation by the use of adhesion molecules, which recognize their counterparts on vascular endothelial cells. CD73 is a lymphocyte differentiation antigen, which has recently been shown to mediate lymphocyte binding to cultured endothelial cells. Here, we have examined its expression and function in inflammatory situations using inflammatory skin diseases as a model. In several idiopathic and allergic disorders of the skin, a vast majority of the skin‐infiltrating lymphocytes were found to express CD73. However, on the circulating lymphocytes of these patients the expression of CD73 does not differ from that of healthy individuals. Of the peripheral blood lymphocytes (PEL) of patients, 13% are CD73+; of these, 9% express the cutaneous lymphocyte antigen (CLA), 32% express CD45RO, and 86% are L‐selectin+. Only 1% of PEL express both CLA and CD73. In contrast, most skin‐infiltrating lymphocytes express both molecules, which led us to investigate the role of CD73 in the skin‐homing behavior of these cells. In the frozen‐section adhesion assay, when PBL were treated with the anti‐CD73 monoclonal antibody 4G4, their binding to the vascular endothelium in inflamed skin was inhibited by 70%. Taken together, our results demonstrate that CD73+ lymphocytes preferentially accumulate into inflamed skin and, most importantly, that CD73 is involved in lymphocyte binding to vessels in inflamed skin. In the future, these findings may offer new means to treat inflammatory disorders of the skin.
A critical role for interleukin‐1 receptor accessory protein in interleukin‐1 signaling
Authors: Korherr Christian , Hofmeister Robert , Wesche Holger , Falk Werner
Published: March 2007

Interleukin‐1 (IL‐1) is a central molecule in inflammation and immune responses whose pleiotropic activities are mediated by the type I IL‐1 receptor (IL‐1RI). The IL‐1RI alone on the cell surface is silent after binding of the ligand. We show that the recently identified IL‐1RI accessory protein (IL‐1RAcP) converts the silent into a fully functional IL‐1RI complex. Although transfection of IL‐1RAcP into IL‐1RAcP‐deficient EL4D6/76 cells did not alter the binding kinetics or dissociation constants of the 125I‐labeled IL‐1α/IL‐1RI complex, a very early event, internalization of the activated receptor complex, and a late event, IL‐1‐stimulated IL‐2 production, were successfully restored. Therefore, recruitment of IL‐1RAcP is a critical early step in the signaling cascade mediated by the IL‐1RI activation complex.
Constitutive macropinocytosis allows TAP‐dependent major histocompatibility compex class I presentation of exogenous soluble antigen by bone marrow‐derived dendritic cells
Authors: Norbury Christopher C. , Chambers Benedict J. , Prescott Alan R. , Ljunggren Hans‐Gustaf , Watts Colin
Published: March 2007

Dendritic cells expanded from mouse bone marrow (BMDC) with granulocyte/macrophage‐colony‐stimulating factor have potent T cell‐stimulatory properties both in vitro and in vivo. This has been well documented for major histocompatibility complex (MHC) class II‐restricted responses, and more recently using peptide‐loaded and protein‐pulsed DC for CD8 responses following adoptive transfer in mice. An unresolved question concerns the capacity of BMDC to present exogenous antigen on MHC class I molecules, an unconventional mode of MHC class I loading for which there is now considerable evidence, particularly in macrophages. Here, we show that BMDC exhibit high levels of macropinocytosis driven by constitutive membrane ruffling activity. Up to one‐third of actively ruffling and macropinocytosing BMDC transferred pinocytosed horseradish peroxidase into the cytosol following a 15‐min pulse, suggesting that they might be capable of presenting exogenous soluble antigen on MHC class I molecules. We show that BMDC presented exogenous ovalbumin to a T cell hybridoma more effectively, more rapidly, and at lower exogenous antigen concentrations than BM macrophages on a cell‐for‐cell basis. Presentation was TAP dependent, brefeldin A sensitive, and blocked by inhibitors of proteasomal processing, demonstrating use of the classical MHC class I pathway. Although effective presentation of exogenous antigen by BMDC occurred in the absence of agents which stimulate macropinocytosis, treatment with phorbol myristate acetate (PMA) enhanced both pinocytosis and MHC class I presentation by BMDC. Finally, PMA‐stimulated BMDC exposed to exogenous ovalbumin in vitro were able to prime an antigen‐specific cytotoxic T lymphocyte response following adoptive transfer in vivo.
Targeted disruption of the VH 81X gene: Influence on the B cell repertoire
Authors: Huetz François , Tornberg Ulla‐Carin , Malanchère Evelyne , Roes Jürgen , Carlsson Leif , Coutinho Antonio , Holmberg Dan , Rajewsky Klaus
Published: March 2007

We have generated a mutant mouse in which the most D‐proximal VH gene (VH81X) has been disrupted by introducing a neomycin‐resistance gene into the VH81X exon by means of gene targeting in embryonic stem cells. The mutant mice generated are unable to express the VH81X gene but appear to display a normal pattern of B cell differentiation as well as normal numbers of bone marrow and peripheral B cells from fetal life all through ontogeny. They mount normal immune responses to several different antigens tested. In contrast, the distribution of VH gene rearrangements in the VH7183 family is altered in homozygous mutant mice. Thus, the antibody repertoire of the targeted mice is modified, at least as far as the expression of VH7183 genes is concerned.
Early B cell factor binds to a site critical for λ5 core enhancer activity
Authors: Mårtensson Annica , Mårtensson Inga‐Lill
Published: March 2007

The pre‐B cell‐specific expression of the λ5 gene is regulated at the level of transcription. The 5′ region of the λ5 gene has been shown to contain an enhancer that activates heterologous promoters. Here, we show that this enhancer, Bλ5, also acts as a lineage‐ and tissue‐restricted enhancer on its own promoter. We define the enhancer core, bλ5, that carries around 50% of the total enhancer activity. We also demonstrate that the transcription factor early B cell factor (EBF) binds to a DNA motif in the λ5 core enhancer which is crucial for enhancer activity, suggesting that λ5 is a second target gene of EBF.
The proteasome‐specific inhibitor lactacystin blocks presentation of cytotoxic T lymphocyte epitopes in human and murine cells
Authors: Cerundolo Vincenzo , Benham Adam , Braud Veronique , Mukherjee Siddhartha , Gould Keith , Macino Beatrice , Neefjes Jacques , Townsend Alain
Published: March 2007

We describe the effect of the proteasome specific inhibitor lactacystin on the metabolic stability of influenza nucleoprotein (NP) and on the generation of antigens presented by human and murine class I molecules of the major histocompatibility complex to cytotoxic T lymphocytes (CTL). We show that cells treated with lactacystin fail to present influenza antigens to influenza‐specific CTL, but retain the capacity to present defined epitopes expressed as peptides intracellularly by recombinant vaccinia viruses. This block in antigen presentation can be overcome by expressing the viral protein within the lumen of the endoplasmic reticulum, confirming the specificity of lactacystin for cytosolic proteases. We also show that the effect of lactacystin on antigen presentation correlates with the block of breakdown of a rapidly degraded form of the influenza NP linked to ubiquitin. These results demonstrate that proteasome‐dependent degradation plays an important role in the cytosolic generation of CTL epitopes.