bluuebunny/crossref_metadata_2025 huggingface dataset:
CD56bright natural killer cell subsets: Characterization of distinct functional responses to interleukin‐2 and the c‐kit ligand
Authors: Carson William E. , Fehniger Todd A. , Caligiuri Michael A.
Published: March 2007

Natural killer (NK) cells are bone marrow‐derived large granular lymphocytes that express the CD56 surface antigen. The CD56bright NK subset represents approximately 10 % of all NK cells and is thought to be the least differentiated NK cell component in blood. The most mature NK cell expresses CD56 at low density and CD16 (FcRγIII) at high density, whereas CD56bright NK cells either lack CD 16 (CD56brightCD16−) or express it at low density (CD56brightCD16dim). c‐kit is a tyrosine kinase receptor which is expressed on both CD34+ hemato‐poietic precursor cells and CD56bright NK cells. In the current study, we characterize interleukin (IL)‐2 receptor (IL‐2R) and c‐kit expression in each of the CD56bright subsets. Both the CD56brightCD16− and CD56brightCD16dim NK subsets express the high‐affinity IL‐2R and the c‐kit receptor when isolated from fresh blood. However, each CD56bright NK cell subset has distinct functional responses to IL‐2, the c‐kit ligand (KL), or both. Activation of the high‐affinity IL‐2R on CD56brightCD16− NK cells induces a proliferative response that is significantly weaker than that observed in the CD56brightCD16dim NK cell subset. Incubation of the CD56brightCD16− NK cell subset with KL significantly enhances IL‐2‐induced proliferation, while KL has no such effect on the CD56brightCD16dim NK subset. Activation of the high‐affinity IL‐2R in both CD56bright subsets induces lymphokine‐activated killer (LAK) activity, but the addition of KL has no effect on LAK activity. Co‐stimulation of either CD56bright subset with IL‐12 and concentrations of IL‐2 that only saturate the high‐affinity IL‐2R induces substantial interferon (IFN)‐γ production. The addition of KL to this co‐stimulatory signal enhances IFN‐γ production in both CD56bright NK subsets. The distinct functional responses to IL‐2 and KL seen in the CD56brightCD16− and CD56brightCD16dim NK subsets provide insight into IL‐2R signaling and suggest that each phenotype identifies a discrete stage of NK cell differentiation.
Targeted elimination of cells expressing the high‐affinity receptor for IgE (FcϵRI) by a Pseudomonas exotoxin‐based chimeric protein
Authors: Fishman Ala , Lorberboum‐Galski Haya
Published: March 2007

The interaction between IgE and its high‐affinity receptor FcϵRI found on mast cells and basophils is the primary effector pathway in allergic response. To achieve a targeted elimination of cells expressing FcϵRI receptors, we constructed a chimeric protein in which a Fc fragment of mouse IgE is attached to a truncated form of Pseudomonas exotoxin (PE). To prepare the targeting moiety, we used a DNA sequence corresponding to amino acids 301–437, representing 30 residues of domain 2 and domain 3 of the mouse IgE constant region. This sequence was fused at the 5′ of a cDNA encoding PE40, a truncated form of PE lacking the cell binding domain. The chimeric protein, termed Fc2′−3−PE40, was expressed in Escherichia coli and partially purified. The protein is highly cytotoxic to mouse mast cell lines and bone marrow‐derived primary mast cells. This cytotoxicity is specific, as it could be blocked upon addition of whole IgE. Moreover, the protein had no effect on other cell lines of hemopoietic origin. The Fc2′−3−PE40 chimeric protein offers a new approach to the treatment of allergic disorders.
CD8 T cells from major histocompatibility complex class II‐deficient mice respond vigorously to class II molecules in a primary mixed lymphocyte reaction
Authors: Shimizu Takeyuki , Takeda Shunichi
Published: March 2007

Mature CD4+ and CD8+ T cells are restricted by major histocompatibility complex (MHC) class II and class I molecules, respectively. In a primary mixed lymphocyte reaction (MLR), CD8+ T cells from C57BL/6 (B6) mice can respond to allo‐class I molecules, but not allo‐class II molecules. However, a significant fraction of CD8+ T cells from C57BL/6 class II‐deficient (B6Aα−) mice violate this rule by responding vigorously in a MLR to class II molecules. The frequency of responding cells is ∼ 50 % of that of B6 CD8+ T cells responding to B6bm1 allo‐class I molecules. This response requires neither appropriate co‐receptor, i.e. CD4, nor exogenous lymphokines, indicating that interactions between the T cell receptors (TCR) and class II molecules are remarkably efficient. Since these CD8+ T cells are positively selected by class I molecules in the thymus of class II‐deficient mice, these CD8+ T cells should interact with both classes of MHC molecules. The absence of thymic negative selection by class II molecules may result in the production of these CD8+ T cells. The data imply that a substantial fraction of CD4+CD8+ double‐positive thymocytes in wild‐type mice interacts with both classes of MHC molecules prior to thymic selection.
Studies in vitro on the mechanism of the epithelial/mesenchymal interaction in the early fetal thymus
Authors: Shinohara Takashi , Honjo Tasuku
Published: March 2007

The effect of the mesenchyme on early thymus development was investigated in vitro by culturing tissue recombinants of the epithelium and mesenchyme derived from the earliest fetal thymus primordium. The thymus mesenchyme promoted the development of the epithelium as assessed by expression of epithelial surface molecules such as major histocompatibility complex (MHC) class I and class II antigens; mesenchymes of other organs were similarly effective. We looked for the culture conditions in which the epithelium could normally express the MHC class II antigen without the mesenchyme and found that the insulin‐like growth factors — I and — II were able to support epithelial development under serum‐free conditions. When we cultured the prospective thymus region of a day 9 embryo which had not initiated lymphoid precursor migration, MHC class II antigen was expressed on the epithelium, indicating that lymphoid precursors are not required for early epithelial differentiation. This system provides a means to dissect the complex tissue interactions during the earliest stages of thymus development.
Function of CD23 in the response of human B cells to antigen
Authors: Reljić Rajko , Cosentino Giovanna , Gould Hannah J.
Published: March 2007

Co‐ligation of antigen receptor and complement receptor 2 (CD21) in the B cell membrane is important in the immune response to T‐dependent antigens. Four CD21 ligands have so far been identified, but only the activated products of the third component of complement (C3) are known to augment the immune response to specific antigens. The most recently discovered ligand for CD21 is CD23. We have generated a CD32+ CD23+ fibroblast cell line which presents a surrogate antigen (anti‐IgM) to human tonsil B cells in vitro. Incubation with these cells causes a 10‐ to 100‐fold reduction in the threshold concentration of anti‐IgM required for B cell proliferation. Anti‐CD19 further enhances the response to antigen and induces proliferation in the absence of anti‐IgM. Addition of soluble CD21 totally inhibits the effect of CD23, suggesting that CD21 mediates synergistic signaling by CD23.
Cytokine stimulation of T lymphocytes regulates their capacity to induce monocyte production of tumor necrosis factor‐α, but not interleukin‐10: Possible relevance to pathophysiology of rheumatoid arthritis
Authors: Sebbag Mireille , Parry Sarah L. , Brennan Fionula M. , Feldmann Marc
Published: March 2007

Previous studies in the laboratory have shown that the pro‐inflammatory cytokine tumor necrosis factor (TNF)‐α plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). The mechanisms involved in regulating monocyte/macrophage cytokine production are not yet fully understood, but are thought to involve both soluble factors and cell/cell contact with other cell types. We and others have previously demonstrated that T cells activated through the T cell receptor/CD3 complex induce monocyte TNF‐α production by contact‐mediated signals. In this report, we investigated further whether T cells activated by cytokines in the absence of T cell receptor stimulation also regulate monocyte cytokine production. T cells were activated in an antigen‐independent manner using the cytokines interleukin (IL)‐15 or IL‐2 alone, or in combination with IL‐6 and TNF‐α. Subsequently, T cells were fixed and incubated with monocytes. Fixed, cytokine‐stimulated T cells induced monocytes to secrete TNF‐α in a dose‐dependent manner, but did not induce secretion of IL‐10, a potent endogenous down‐regulator of TNF‐α and other pro‐inflammatory cytokines. Stimulation of monocyte TNF‐α was markedly inhibited when T cells were physically separated from monocytes within the tissue culture well, confirming that T cell contact is necessary. T cell acquisition of monocyte‐activating capacity was shown to be dependent on the period of cytokine stimulation, with T cells activated for 8 days more effective than T cells activated for shorter periods. Addition of interferon‐γ or granulocyte/macrophage colony‐stimulating factor to the T cell/monocyte cultures enhanced T cell induction of monocyte TNF‐α by threefold and ninefold, respectively. The results from this model of cognate interaction suggest that cytokine‐stimulated T cells, interacting with macrophages in the rheumatoid synovial membrane, may contribute to the continuous excessive production of TNF‐α observed in the RA joint, and to the imbalance of pro‐inflammatory cytokines over anti‐inflammatory cytokines.
Interleukin‐7 activates human naive CD4+ cells and primes for interleukin‐4 production
Authors: Webb Louise M. C. , Foxwell Brian M. , Feldmann Marc
Published: March 2007

Interleukin (IL)‐4 is considered to be essential for T helper (Th)2 cell development, yet in areas of primary T cell activation, CD4+ cells are its only source. This implies that other signals must drive the initial expression of IL‐4 production. The role of CD28 co‐stimulation in Th2 subset development has been described. However, in mice deficient for CD28, Th2 responses are diminished, but not abrogated. Cytokines produced within the lymphoid tissue, e.g. IL‐7, may be important in the primary activation of naive CD4+ cells. We have found that human naive CD4+ cells purified from umbilical cord blood express the IL‐7 receptor and respond vigorously to IL‐7 during primary stimulation. Naive CD4+ cells grown in IL‐4, in the presence or absence of IL‐2, fail to produce Th2 cytokines upon restimulation. In contrast, IL‐7 induces development of a population of T cells that produce large amounts of IL‐4. Growth in IL‐7 also increases IL‐2‐induced production of interferon (IFN)‐γ and IL‐10 production. IL‐7‐induced IL‐4 production is not inhibited by neutralizing antibodies to IL‐4 on its receptor. This implies that IL‐7 acts directly to induce Th2 subset development and not by up‐regulating either production of IL‐4 during culture or expression of the IL‐4 receptor. Moreover, IL‐7 potentiates the effects of CD28 co‐stimulation on both naive CD4+ cell proliferation and subsequent IL‐4 production. Following primary stimulation, CD4+ cells lose expression of the IL‐7 receptor, resulting in IL‐7 unresponsiveness. This work reveals a novel role for IL‐7 in the primary activation of CD4+ cells. We propose that in conjunction with CD28 co‐stimulation, IL‐7 induces the initial expression of IL‐4 production and that IL‐4 acts subsequently to expand Th2 cytokine‐producing cells at the appropriate anatomical site.
Prevention of experimental autoimmune uveoretinitis by monoclonal antibody to interleukin‐12
Authors: Yokoi Hidetoshi , Kato Kazunori , Kezuka Takeshi , Sakai Jun‐Ichi , Usui Masahiko , Yagita Hideo , Okumura Ko
Published: March 2007

Experimental autoimmune uveoretinitis (EAU) induced by immunization with interphotoreceptor retinoid‐binding protein (IRBP), a retinal self antigen, has been regarded to be a typical T helper type 1 (Th1)‐mediated inflammatory disease. In this study, we examined the effect of a neutralizing monoclonal antibody (mAb) to interleukin‐12 (IL‐12), which has been known to play a critical role in the Th1 differentiation, on the development of EAU. While 9 of 13 control mice developed EAU by the immunization with IRBP, none of 12 mice developed EAU when given anti‐IL‐12 mAb 1 day before immunization. These mice did not develop EAU even after a rechallenge with IRBP on day 30, indicating that a protective mechanism had been established by the anti‐IL‐12 treatment. The proliferative response of splenocytes to IRBP in vitro was not significantly impaired, but the production of IL‐2 and IFN‐γ was greatly reduced by the anti‐IL‐12 treatment. Moreover, production of IL‐5 and expression of IL‐4 mRNA were increased by the anti‐IL‐12 treatment. Consistently, IgG2a anti‐IRBP serum antibodies were decreased and IgG1 were increased. Administration of a neutralizing anti‐IL‐4 mAb at the time of IRBP rechallenge reversed the protection established by the anti‐IL‐12 treatment at the primary immunization. These results indicate that the anti‐IL‐12 treatment at the IRBP priming not only prevented the development of pathogenic Th1 cells, but also induced suppressive Th2 cells that protect the animals from further challenge with the same antigen.
Thymocyte activation induces the association of phosphatidylinositol 3‐kinase and pp120 with CD5
Authors: Dennehy Kevin M. , Broszeit Richard , Garnett David , Durrheim Glenda A. , Spruyt Louise L. , Beyers Albert D.
Published: March 2007

CD5 is a glycoprotein expressed on thymocytes, T cells, and a subset of B cells. Antibody‐mediated cross‐linking studies or studies on CD5 knockout mice implicate CD5 as a co‐stimulatory or negative regulatory molecule. CD5 is rapidly phosphorylated on tyrosine (Y) residues following T cell activation. Y429 and Y441 occur in an imperfect immunoreceptor tyrosine‐based activation motif (ITAM)‐like sequence. We investigated whether phosphatidylinositol (PI) 3‐kinase, which binds to tyrosine‐phosphorylated ITAM, interacts with CD5 following T cell activation. PI 3‐kinase activity and the regulatory p85 subunit of PI 3‐kinase associated with CD5 in pervanadate‐stimulated, but not in unstimulated thymocytes. Cellular p85 as well as the recombinant Src homology 2 (SH2) domains of p85 bound a tyrosine‐phosphorylated peptide encompassing Y463 with approximately threefold greater affinity than a doubly tyrosine‐phosphorylated Y429‐Y441 peptide. Binding of the C‐SH2 domain to the Y463 phosphopeptide, together with preferential binding of the N‐SH2 domain to the Y429‐Y441 phosphopeptide, suggests a bivalent interaction. A 120‐kDa phosphoprotein (pp 120) associated with CD5 and specifically with the Y429‐Y441 phosphopeptide in stimulated thymocytes. We conclude that stimulation of thymocytes with pervanadate induces the recruitment of PI 3‐kinase and pp 120 to CD5.
Growth arrest of γδ T cells induced by monoclonal antibody against WC1 correlates with activation of multiple tyrosine phosphatases and dephosphorylation of MAP kinase erk2
Authors: Kirkham Paul A. , Takamatsu Haru‐Hisa , Parkhouse R. Michael E.
Published: March 2007

WC1 is a 215‐kDa type 1 transmembrane glycoprotein, the expression of which is restricted to γδ T lymphocytes. The binding of an anti‐WC1 monoclonal antibody (mAb) (SC‐29) induces reversible growth arrest in proliferating interleukin (IL)‐2‐dependent γδ T lymphocytes and this study has examined the relevant biochemical mechanisms. WC1 binding activates multiple protein tyrosine phosphatases causing specific tyrosine dephosphorylation in the absence of calcium mobilization. One of the dephosphorylated proteins was identified as the MAP kinase erk2. Another phosphotyrosine protein of 70 kDa, found to coprecipitate with p85 phosphoinositol (PI)3‐kinase was either dephosphorylated or uncoupled from the p85 PI 3‐kinase immunoprecipitate after WC1 receptor binding by mAb SC‐29. The anti‐WC1‐induced tyrosine dephosphorylation was reversed by stimulation of γδ T cells with concanavalin A or anti‐CD3 mAb, demonstrating that at the biochemical level, mitogen stimulation is dominant to the growth‐arresting effects of anti‐WC1. It is therefore proposed that the activation of tyrosine phosphatases by WC1 binding and the resultant dephosphorylation of certain key signaling protein such as erk2 correlates with and may cause the induction of growth arrest in IL‐2‐dependent γδ T cells, without affecting the cells ability to respond to antigen. Possible mechanisms, which include the inhibition of IL‐2 signal transduction pathways, are discussed.
Interleukin‐10 prevents the generation of dendritic cells from human peripheral blood mononuclear cells cultured with interleukin‐4 and granulocyte/ macrophage‐colony‐stimulating factor
Authors: Buelens Christel , Verhasselt Valérie , De Groote Donat , Thielemans Kris , Goldman Michel , Willems Fabienne
Published: March 2007

We evaluated the effects of interleukin (IL)‐10 on the differentiation of dendritic cells (DC) obtained by culturing plastic‐adherent peripheral blood mononuclear cells for 7 days in presence of granulocyte/macrophage‐colony‐stimulating factor (GM‐CSF)+IL‐4. The addition of IL‐10 at the initiation of culture resulted in the generation of macrophage‐like cells with expressing high levels of CD14 and decreased levels of CD1a and CD1c. Furthermore, cells generated in presence of IL‐10 secreted lower levels of IL‐12, but higher levels of IL‐8 compared with DC generated in absence of IL‐10, both spontaneously and after CD40 engagement. Finally, cells generated in presence of IL‐10 were less efficient than DC in stimulating the production of IL‐2, interferon‐γ, and IL‐4 by allogeneic T cells. We conclude that IL‐10 prevents the generation of DC induced by GM‐CSF+IL‐4 and favors the development of macrophages with a lower T cell stimulatory potential, but secreting higher levels of IL‐8 than DC.
Interleukin‐10 and interleukin‐4 inhibit intracellular killing of Leishmania infantum and Leishmania major by human macrophages by decreasing nitric oxide generation
Authors: Vouldoukis Ioannis , Bécherel Pierre‐André , Riveros‐Moreno Valentina , Arock Michel , Da Silva Otamires , Debré Patrice , Mazier Dominique , Mossalayi M. Djavad
Published: March 2007

The host response to Leishmania infection is regulated by a specific pattern of local cytokine production. We investigated the effect of interleukin (IL)‐10 and IL‐4 on the leishmanicidal activity of human macrophages (Mϕ). As with L. major, intracellular killing of L. infantum by human Mϕ was obtained following ligation of surface CD23 or cell treatment with Interferon‐γ (IFN‐γ). This leishmanicidal activity required nitric oxide (NO) generation by activated Mϕ, and it was partially mimicked by cell treatment with chemical NO donors. Addition of recombinant human IL‐10 or IL‐4 to CD23 mAb or IFN‐γ decreased L. infantum and L. major killing by infected Mϕ. IL‐10 was more potent than IL‐4 in inhibiting the leishmanicidal activity of human Mϕ. Inhibition of Leishmania killing by IL‐4 and IL‐10 correlated with decreased NO generation from Mϕ, and was reversed when exogenous NO was added to cell cultures. Therefore, IL‐10 and IL‐4 down‐regulate leishmanicidal activity of human Mϕ, in part by inhibiting NO generation by these cells.
Cytotoxicity and weak CD40 ligand expression of CD8+ type 2 cytotoxic T cells restricts their potential B cell helper activity
Authors: Sad Subash , Krishnan Lakshmi , Chris Bleackley R. , Kägi David , Hengartner Hans , Mosmann Tim R.
Published: March 2007

Naive CD8+ T cells differentiate into distinct cytokine‐secreting subsets: T helper (Th)1‐like cytotoxic T cells (Tc1) and Th2‐like Tc2. Although Th2 cells provide strong B cell help, we show that Tc2 cells secreting the same cytokines provide only modest B cell help for IgM production, and only when large numbers of B cells were stimulated with small numbers of Tc2 cells. Lack of effective B cell help by Tc2 cells was attributable partly to their cytotoxicity towards B cells. Both Tc1 and Tc2 cells killed small resting B cells mainly by a perforin‐dependent mechanism. In contrast to normal Tc2 cells, perforin‐deficient Tc2 cells failed to kill small resting B cells and induced IgM and IgG1 production, although their B cell help was significantly lower than that mediated by Th2 cells. This may be partly attributable to the ability of Tc2 but not Th2 cells to kill activated B cells even in the absence of perforin. Plate‐bound anti‐CD3 antibodies inhibited Tc2 killing of B cells and induced substantial immunoglobulin production. Additionally, Tc1 and Tc2 cells failed to express CD40 ligand (CD40L), whereas Th1 and Th2 cells expressed high levels of CD40L. Stimulation of Tc1 and Tc2 cells with plate‐bound anti‐CD3 antibodies for extended periods resulted in low‐level expression of CD40L. Proliferation of small resting B cells correlated with immunoglobulin production: proliferation was promoted strongly by Th1 and Th2, weakly by normal Tc1 and Tc2, and moderately by perforin‐deficient Tc1 and Tc2 cells. Thus, Tc2 cells may not contribute significantly to cognate B cell help during normal responses.
Expression of killer cell inhibitory receptors on human uterine natural killer cells
Authors: Verma Sanjay , King Ashley , Loke Yung Wai
Published: March 2007

The establishment of the human placenta in early pregnancy is characterized by the presence of large numbers of natural killer (NK) cells within the maternal decidua in close proximity to the fetally‐derived invading extravillous trophoblast which expresses at least two HLA class I molecules, HLA‐G and HLA‐C. These NK cells have an unusual phenotype, CD56bright CD16, distinguishing them from adult peripheral blood NK cells. They may control key events in trophoblast migration and therefore placentation. Human NK cells in peripheral blood express receptors for polymorphic HLA class I molecules. This family of receptors, known as killer cell inhibitory receptors (KIR), are expressed on overlapping subsets of NK cells to give an NK cell repertoire which differs between individuals. Using a panel of monoclonal antibodies to several members of the KIR family and analysis by flow cytometry, we have found that KIR are expressed by decidual NK cells. There is variation in both the percentage of cells expressing a particular receptor and the density of receptor expression between decidual NK cells from different individuals. Comparison of NK cells from decidua and peripheral blood of the same individual showed that NK cells from these two different locations express different repertoires of KIR. Receptors are present in individuals who do not possess the relevant class I ligand, raising the possibility that these NK receptors may be involved in recognition of the allogeneic fetus by the mother at the implantation site.
Up‐regulation of human epidermal Langerhans' cell B7‐1 and B7‐2 co‐stimulatory molecules in vivo by solar‐simulating irradiation
Authors: Laihia Jarmo K. , Jansen Christer T.
Published: March 2007

Ultraviolet (UV) radiation impairs cutaneous immune functions and induces antigen‐specific tolerance both locally at the irradiated skin site, as well as at distant skin sites and systemically. It has been postulated that in the local model, altered Langerhans' cells (LC) provide tolerogenic signals, and studies in vitro have indicated that UV radiation may down‐regulate the expression of co‐stimulatory molecules on the surface of these cells. To examine the effect of UV radiation on LC co‐stimulatory molecules in vivo, we irradiated human volunteers with erythematogenic doses of solar‐simulating UV radiation (SSR), and analyzed the expression of cell surface markers in dermatome skin samples obtained 1–72 h post‐irradiation. For flow cytometric analysis, epidermal cell (EC) suspensions were prepared and double labeled with monoclonal antibodies against CD1a or HLA‐DR, and B7‐1 (CD80), B7‐2 (CD86), ICAM‐1 (CD54), ICAM‐3 (CD50), LFA‐3 (CD58), E‐cadherin, or integrin‐β4 (CD104). In unirradiated control skin samples, keratinocytes (KC) expressed high levels of E‐cadherin. LC expressed high levels of both E‐cadherin and ICAM‐3, and low levels of B7‐2, LFA‐3, ICAM‐1, and integrin‐β4. Following SSR, a triphasic reaction pattern was seen: an immediate, down‐regulatory phase prevailing 2–6 h post‐irradiation, when the number of DR+ and CD1a+ cells were temporarily reduced; a delayed, up‐regulatory phase in which the number of LC was increased and the expression intensities of CD1a, HLA‐DR, B7‐1, and B7‐2 were strongly up‐regulated, maximally evident 12–24 h after irradiation, but no more seen at 48 h; and a late phase at 72 h, in which an influx of monocytes and a concomitant rise in DR+ cells was recorded. We conclude that to understand real‐life cutaneous UV immunology, studies in vitro need to be complemented with studies in vivo. In the case of LC, the effects of erythematogenic UV radiation in vivo on human LC B7 co‐stimulatory molecules include an up‐regulatory stage.
Deregulated TCRαβ T cell population provokes extramedullary hematopoiesis in mice deficient in the common γ chain
Authors: Sharara Lama I. , Andersson ÅSa , Guy‐Grand Delphine , Fischer Alain , Disanto James P.
Published: March 2007

Deficiency of the cytokine receptor common γ chain (γc) results in abnormal lymphoid development and a severe immunodeficiency disease due to the combined loss of the receptors for interleukins (IL)‐2, ‐4, ‐7, ‐9, and ‐15. We have observed the development of secondary hematopoiesis with circulating hematopoietic progenitor cells in adult mice harboring a null mutation in γc. These extramedullary changes were not secondary to bone marrow failure or to an inability to maintain circulating blood counts. These results suggested that γc‐dependent cytokine signaling pathways modulate hematopoietic development. An intrinsic defect in γc− hematopoietic stem cell committment appeared unlikely, as fetal liver hematopoiesis was unaltered in γc− embryos. Furthermore, the absence of natural killer cells in γc− mice was not responsible for the observed hematopoietic changes. Peripheral TCRαβ T cells from γc− mice were characterized by an activated phenotype (CD62Llo, CD44hi, CD69hi) and showed increased levels of transcripts for hematopoietic stimulating cytokines, including IL‐3 and granulocyte/macrophage‐colony‐stimulating factor. A predominance of these cells was detected in the bone marrow, suggesting a role for residual T cells in the enhanced hematopoiesis. Strikingly, the elimination of residual T cells from γc− mice reduced splenic and circulating hematopoietic precursor frequencies to normal levels. These results clearly implicate a deregulated TCRαβ T cell population in the observed hematopoietic changes in γc− mice, and emphasize the importance of γc‐dependent cytokine interactions in modulating mature T cell responses.
Mice heterozygous for a deletion of the tumor necrosis factor‐α and lymphotoxin‐α genes: biological importance of a nonlinear response of tumor necrosis factor‐α to gene dosage
Authors: Amiot Franck , Boussadia Oréda , Cases Sylvaine , Fitting Catherine , Lebastard Mai , Cavaillon Jean‐Marc , Milon Geneviève , Dautry François
Published: March 2007

The tumor necrosis factors (TNF‐α and lymphotoxin, or LT‐α) are important mediators of the immune and inflammatory responses, and it has been proposed that a positive feedback loop could boost the expression of the TNF to sufficiently high levels to fend off infections. To investigate this phenomenon and its biological consequences, we have generated LT‐α/TNF‐α knockout mice and compared mice having one or two functional LT‐α/TNF‐α alleles. In response to lipopolysaccharide (LPS) stimulation, TNF‐α levels in the circulation or in the supernatant of macrophage cultures were 20‐ to 100‐fold lower in heterozygous samples than in their wild‐type counterparts. This differential increased with the intensity of stimulation and throughout the response, supporting the involvement of a positive feedback loop. Moreover, the heterozygous mice had an increased bacterial load following Listeria monocytogenes infection and exhibited a bimodal response to the association of D‐galactosamine and LPS which was similar to that of wild‐type mice at low doses of LPS and more like that of homozygous mutants at high doses. These results therefore establish the biological importance of the nonlinear response of TNF‐α levels to gene dosage, and these mice provide a unique tool to study how the propensity to produce TNF can determine the immunological fitness of individuals.
Insect venom immunotherapy induces interleukin‐10 production and a Th2‐to‐Th1 shift, and changes surface marker expression in venom‐allergic subjects
Authors: Bellinghausen Iris , Metz Gudrun , Enk Alexander H. , Christmann Steffen , Knop JÜRgen , Saloga Joachim
Published: March 2007

The current study was carried out to elucidate the immunoregulatory changes induced by venom immunotherapy (VIT) in bee or wasp allergic subjects. All subjects included in this study had a history of severe systemic allergic reactions to stings of the respective insect as well as positive skin tests with the respective venom or venom‐specific IgE in the sera. Parameters assessed in peripheral blood mononuclear cells (PBMC) before and after initiation of VIT (rush therapy reaching a maintenance dose of 100 μg venom injected subcutaneously within 1 week) were expression of CD3, CD4, CD8, CD45RA, CD45R0, interleukin (IL)‐2 receptor (R)α, IL‐4R, IL‐12R, FcσRII, CD40, and CD40 ligand (CD40L), cells producing interferon (IFN)‐γ and IL‐10 after stimulation with phorbol 12‐myristate 13‐acetate + ionomycin in the presence of monensin measured by flow cytometry; secretion of IFN‐γ, IL‐4, and IL‐10 measured by ELISA (IFN‐γ and IL‐10 were additionally measured by PCR), and proliferation after stimulation with the respective venom. Significant decreases were observed after VIT for proliferative response to venom and venom + IL‐4, IL‐4 secretion, FcσRII, CD40, and CD40L expression. Significant increases were observed after VIT for IFN‐γ concerning the amount secreted and the number of producing cells, and IL‐10. IL‐10 was mainly produced by CD4+ cells that were negative for IFN‐γ, but some double‐positive (IL‐10 and IFN‐γ) cells were always detected. Addition of blocking anti‐IL‐10 antibodies, but not isotype control antibodies, prevented down‐regulation of proliferation (but not IL‐4 secretion) and further enhanced IFN‐γ secretion after VIT. These data indicate that in insect venom allergic subjects, VIT not only induces a rapid shift in cytokine expression from Th2 to Th1 cytokines, but also leads to induction of the immunosuppressive cytokine IL‐10, which may be important for the limitation of potentially harmful allergen‐specific Th1 responses. The described changes in cytokine expression may be responsible for subsequent increases in allergen‐specific IgG and decreases in IgE production, as well as suppressive activity observed in earlier studies.
The human major histocompatibility complex class Ib molecule HLA‐E binds signal sequence‐derived peptides with primary anchor residues at positions 2 and 9
Authors: Braud Veronique , Yvonne Jones E. , McMichael Andrew
Published: March 2007

Human histocompatibility leukocyte antigen E (HLA‐E) and mouse major histocompatibility complex (MHC) class Ib antigen, Qa‐1, share the same substitutions at two normally conserved positions 143 and 147, which are likely to affect binding of the C terminus of peptides. Qa‐1 is able to bind a peptide derived from the leader sequence of H‐2 D and H‐2 L molecules. We developed a peptide binding assay in vitro to compare the binding specificity of HLA‐E with the mouse MHC class Ib molecule Qa‐1. We demonstrate that HLA‐E binds, although poorly, the peptide which binds to Qa‐1 and that it also binds nonamer signal sequence‐derived peptides from human MHC class I molecules. Using alanine and glycine substitutions, we could define primary anchor residues at positions 2 and 9 and secondary anchor residues at position 7 and possibly 3.
Stringent thiol‐mediated retention in B lymphocytes and Xenopus oocytes correlates with inefficient IgM polymerization
Authors: Carelli Stephana , Ceriotti Aldo , Sitia Roberto
Published: March 2007

Thiol‐dependent retention mechanisms involving the μs chain Cys575 ensure that only polymeric IgM are secreted. B lymphocytes are unable to polymerize IgM and degrade unpolymerized precursors intracellularly. Since several non‐lymphoid transfectants secrete hexameric IgM, specific mechanism(s) inhibiting IgM polymerization/secretion may be active in B cells. Here, we show that Xenopus laevis oocytes are also unable to polymerize IgM and retain this isotype via Cys575 as efficiently as B cells. The mechanisms and the hierarchy of the thiol‐dependent pre‐Golgi retention are conserved in amphibian oocytes, as indicated by the efficient retention of secretory IgA and the slow secretion of unassembled J558 λ chains. We also show that B cells do not lack any structural component necessary to polymerize IgM: after retention has been weakened by 2‐mercaptoethanol, polymerization can occur if oxidizing conditions are restored. Since release from retention can result in polymerization, stringent retention in B cells and oocytes might be at the basis of their common inability to polymerize secretory IgM. Our findings suggest that disulfide interchange reactions in the exocytic compartment can be modulated during B cell differentiation to control IgM secretion.
Diverse transcription factors are involved in the quantitative regulation of transcriptional activation of χ promoters
Authors: Bemark Mats , Leanderson Tomas
Published: March 2007

Immunoglobulin χ promoters show sequence divergence but conserved function between different subgroups. Here we show that three separate 5′ elements are required for synergistic stimulation of transcription with the decamer in a χ promoter. These sites are a 5′ E‐box, a 3′ AT‐rich region in the pentadecamer (pd) element, and the χ‐Y element. Elf‐1 is a novel χ‐Y element ligand induced upon mitogenic stimulation of resting B lymphocytes. Furthermore, the 5′ E2A‐like E‐box in the pd element could be substituted by an upstream stimulatory factor motif with conservation of function. Thus, the synergistic activation requirements of χ transcription is strictly dependent on the quantitative presence of transcription factor‐binding motifs 5′ of the decamer, but these differ qualitatively in that they may bind an array of proteins with conserved function.
Induction of CD4+ T cell depletion in mice doubly transgenic for HIV gp120 and human CD4
Authors: Finco Oretta , Nuti Sandra , de Magistris Maria Teresa , Mangiavacchi Lucia , Aiuti Alessandro , Forte Pietro , Fantoni Antonio , Putten Herman Van Der , Abrignani Sergio
Published: March 2007

It has been suggested that loss of uninfected T cells in HIV infection occurs because of lymphocyte activation resulting in cell death by apoptosis. To address the question of whether cross‐linking of CD4/HIV gp120 complexes by antibodies were sufficient to induce T cell depletion in vivo, we developed an animal model of continuous interaction between human CD4 (hCD4), gp120 and anti‐gp120 antibodies in the absence of other viral factors. Double‐transgenic mice have been generated in which T cells express on their membrane hCD4 and secrete HIV gp120. Although these mice have hCD4/gp120 complexes present on the surface of T cells, they do not show gross immunological abnormalities, and they are able to produce anti‐gp120 antibodies following immunization with denaturated gp120. However, double‐transgenic mice with antibodies to gp120, when immunized with tetanus toxoid, mount an IgG response that is significantly lower than that of double‐transgenic mice without antibodies to gp120. Furthermore, the presence of anti‐gp120 antibodies leads to CD4+ T cell depletion and immunodeficiency in the absence of HIV infection. Thus, the antibody response to gp120 can lead to CD4+ T cell attrition in vivo.
HTLV‐I‐infected T cells activate autologous CD4+ T cells susceptible to HTLV‐I infection in a co‐stimulatory molecule‐dependent fashion
Authors: Takamoto Tetsuro , Makino Masahiko , Azuma Miyuki , Kanzaki Tamotsu , Baba Masanori , Sonoda Shunro
Published: March 2007

A vigorous production of human T lymphotropic virus type I (HTLV‐I)‐infected CD4+ T cells is closely associated with the development of adult T cell leukemia (ATL) and neurological disease. However, the immunological mechanisms leading to generation of the HTLV‐I‐infected cells are not fully clarified. The modulation of CD80 and CD86 expression on the HTLV‐I‐infected cells and its physiological role in the interaction of infected CD4+ T cells with uninfected CD4+ T cells was examined. The HTLV‐I‐infected CD4+ T cell lines established from ATL patients and normal donors by infecting their CD4+ T cells with the virus expressed CD80, CD86, and HLA‐DR, and induced a proliferation of autologous and allogenic CD4+ T cells. While the CD4+ T cells stimulated with the autologous HTLV‐I‐infected cells for 7 days expressed CD80 and CD86 but not HTLV‐I gene products, they expressed HTLV‐I gag antigen after 4 weeks. The interaction of HTLV‐I‐infected and ‐uninfected CD4+ T cells was profoundly suppressed by a combination of CD80 and CD86 monoclonal antibodies. These results suggest that the induction of CD80 and CD86 on HTLV‐I‐infected CD4+ T cells participates actively in the generation of the virus‐infected progenitor cells.
Identification of epitopes on a mutant form of Pseudomonas exotoxin using serum from humans treated with Pseudomonas exotoxin containing immunotoxins
Authors: Roscoe Donna M. , Pai Lee H. , Pastan Ira
Published: March 2007

PE38 is a 38‐kDa derivative of the 66‐kDa Pseudomonas exotoxin (PE) in which the cell binding domain of PE (domain Ia, amino acids 1–252) and a portion of domain Ib (amino acids 365–380) are deleted. The immunotoxins LMB‐1 and LMB‐7 contain PE38 and kill cancer cells by exploiting the cytotoxic action of PE38. The major human B cell epitopes of PE38 were mapped by measuring the reactivity of 45 serum samples from patients treated with the PE38‐containing immunotoxins LMB‐1 or LMB‐7 to two panels of overlapping synthetic peptides representing the sequence of PE38. One panel of peptides is ten amino acids long and overlap by seven amino acids, and the second panel of peptides is twenty amino acids long and overlap by ten. Five major epitopes were identified: amino acids 274–283, 470–492, 531–540, 555–564, and the C‐terminal amino acids 596–609. Two minor epitopes were identified as well: amino acids 501–510 and 582–589. These epitopes are predominantly located on the surface of the protein. The amino acids believed to be critical for binding are highly solvent‐accessible residues. The results of the human antibody response to peptides are compared to the pattern of reactivity previously identified with serum samples obtained from monkeys administered LMB‐1 and LMB‐7. The epitopes between monkey and human are almost identical, demonstrating similarity in the response of antibody repertoires between the two species and providing further support that these are the immunodominant epitopes. This information is critical for genetically engineering less immunogenic immunotoxins and provides a foundation for the development of a vaccine against pseudomonal infections which plague immunocompromised individuals and individuals with cystic fibrosis.
The role of E‐ and P‐selectin in neutrophil and monocyte migration in adjuvant‐induced arthritis in the rat
Authors: Walter Ulrike M. , Issekutz Andrew C.
Published: March 2007

The role of the endothelial adhesion molecules E‐ and P‐selectin in leukocyte accumulation in arthritis is not known. We investigated this role in rat adjuvant arthritis by employing adhesion function‐blocking monoclonal antibodies (mAb) to rat P‐ and E‐selectin. The acute migration (2 h) of radiolabeled rat blood neutrophils and monocytes to joints and skin was determined. Anti‐P‐selectin mAb significantly reduced accumulation of monocytes (by 50%) and neutrophils (by 40%) in the talar joint, and of neutrophils in tail joints (by 90%). Anti‐E‐selectin mAb alone did not attenuate leukocyte migration, but when combined with anti‐P‐selectin mAb, it enhanced inhibition of neutrophil accumulation in the talar and carpal joints. In the same animals, anti‐P‐selectin mAb significantly inhibited neutrophil and monocyte migration to dermal inflammatory reactions induced by zymosan‐activated rat serum (ZAS) containing the chemotactic factor C5ades Arg, endotoxin (LPS), interferon‐γ (IFN‐γ) and tumor necrosis factor‐α (TNF‐α). In contrast, anti‐E‐selectin mAb alone had no effect on monocyte or neutrophil accumulation in inflamed skin of arthritic animals, but again enhanced the inhibition when combined with mAb to P‐selectin. The addition of anti‐L‐selectin mAb to anti‐P‐ and E‐selectin mAb did not further suppress monocyte or neutrophil migration to inflamed skin or joints. These results demonstrate that optimal leukocyte migration to arthritic joints and inflamed skin is P‐selectin dependent, and E‐selectin is not essential. However, E‐selectin contributes to migration when P‐selectin mechanisms are not operative. L‐selectin does not play a role in E‐ and P‐selectin‐independent leukocyte migration to joints or skin inflammation in arthritic rats. However, it is likely that additional selectin‐independent pathways also mediate neutrophil and monocyte migration to joint and skin inflammation.
Cytokine responses induced by Toxoplasma gondii in astrocytes and microglial cells
Authors: Fischer Hans‐Georg , Nitzgen Bernd , Reichmann Gaby , Hadding Ulrich
Published: March 2007

To investigate the role of astroglia in intracerebral immune response to Toxoplasma gondii, astrocytes cultured from mouse brain were inoculated with mouse‐virulent or ‐avirulent toxoplasma strains. In comparison to microglia/brain macrophages, astrocytes as host cells allowed stronger proliferation of avirulent parasites. Toxoplasma infection of astroglia was accompanied by release of interleukin‐ (IL)1α, IL‐6, and granulocyte/macrophage colony‐stimulating factor (GM‐CSF) activity, whereas alternative challenge by lipopolysaccharide (LPS) evoked no IL‐1 response and significantly higher titers of IL‐6 and GM‐CSF. At the mRNA level, both stimuli induced transcription of all three cytokines in astrocytes. Secretion of IL‐1 and IL‐6 upon infection was triggered by T. gondii brady‐ and tachyzoites in a time‐ and dose‐dependent manner. Heat killing of parasites, but not an exposure to polymyxin B, abrogated their cytokine‐inducing activity, thus indicating that an LPS‐independent stimulus is provided by T. gondii. When administered in combination, LPS synergistically augmented the IL‐1‐inducing effect of toxoplasma infection. In comparison, T. gondii‐induced, but not an LPS‐triggered, IL‐6 response of astrocytes resisted to antagonization with IL‐10. The IL‐6 response of parasitized astroglia was up‐regulated by external tumor necrosis factor (TNF)‐α and transforming growth factor (TGF)‐β1, with only TNF‐α enhancing simultaneous release of IL‐1. Substantial secretion of IL‐10 and TNF‐α was detected in T. gondii‐infected microglia, but not in astrocyte cultures. A possibly autocrine stimulation of infected astroglia via IL‐1 was found to be unlikely, since addition of IL‐1 receptor antagonist did not affect the release of IL‐6 and GM‐CSF while inhibiting these responses in IL‐1‐treated cells. The findings substantiate a separate, T. gondii‐induced pathway of astroglia activation characterized by the release of IL‐1 which may drive local inflammatory reaction both at initial infection of the brain and during reactivating toxoplasmosis.
Prevention of endotoxin‐induced lethality in neonatal mice by interleukin‐13
Authors: Nicoletti Ferdinando , Mancuso Giuseppe , Cusumano Vitaliano , Marco Roberto Di , Zaccone Paola , Bendtzen Klaus , Teti Giuseppe
Published: March 2007

Interleukin(IL)‐13, a cytokine produced by T helper 2 (Th2) cells, is a powerful inhibitor of macrophage functions, including surface expression of CD14 and production of IL‐1 and tumor necrosis factor (TNF)‐α. We tested the effects of recombinant mouse(m)IL‐13 in a neonatal mouse model of endotoxin shock; this is a macrophage‐dependent condition, which is a model of neonatal sepsis in humans. mIL‐13 (0.5 μg/mouse) dramatically reduced the lethal effects of lipopolysaccharide (LPS) if administered either 24 or 4 h prior to or concomitantly with LPS challenge. This action might be mediated by multiple modulatory activities of IL‐13 on LPS‐induced cytokine secretion since, relative to control animals, the mice treated with mIL‐13 had eight times lower peak blood levels of TNF. The IL‐1β levels were also decreased, whereas increased levels of IL‐6 and IL‐10 were observed at several time points after LPS challenge.
An amphibian CD3 homologue of the mammalian CD3 γ and δ genes
Authors: Dzialo Robin C. , Cooper Max D.
Published: March 2007

T cell receptor (TCR) genes have been identified in representatives of both cartilaginous and bony vertebrates. The CD3 chains that serve as signal transducing elements of the TCR complex in mammals have been defined to a limited extent in birds. In these studies a CD3 homologue was identified in an amphibian representative, Xenopus laevis, using degenerate oligomer primers designed from conserved regions of avian and mammalian CD3γ/δ subunits. The reverse transcriptase polymerase chain reaction amplified product of Xenopus splenocyte RNA was then used to isolate full‐length cDNA clones from a splenic library. When employed as probes, the cDNA clones hybridized with a 1‐kb mRNA transcript in Xenopus T cells, but not in other cell types. Comparison of the deduced amino acid sequence indicated a similar degree of homology with mammalian and avian CD3 γ and δ chains. Genomic analysis indicated that the Xenopus CD3 molecule is encoded by five exons, a structure resembling the mammalian CD3δ gene rather than the seven exon CD3γ gene. Southern blot analysis and sequencing of the 5′ flanking region failed to yield evidence of a related Xenopus gene. This amphibian CD3 gene thus appears to represent an ancestral form of the mammalian CD3 γ and δ genes.
Antigen‐specific T cell receptor antagonism by antigen‐presenting cells treated with the hemolysin of Listeria monocytogenes: a novel type of immune escape
Authors: Darji Ayub , Stockinger Brigitta , Wehland Jürgen , Chakraborty Trinad , Weiss Siegfried
Published: March 2007

We have examined the influence of listeriolysin O (LLO), the hemolysin secreted by the pathogenic bacterium Listeria monocytogenes, on major histocompatibility complex class II‐dependent T cell activation. Stimulation of T cells by native antigens but not by peptides is inhibited upon pretreatment of antigen‐presenting cells (APC) with LLO. Experiments presented here reveal that this inhibition is not due to a lack in processing of antigen by APC but is the result of an irreversible inactivation of T cells that recognize antigen on LLO‐treated APC. Incubation of mixtures of two different T cells where only one antigen was presented on LLO‐treated APC suggested that T cell inactivation is antigen specific. The inactivation was dominant and could be observed even in the presence of amounts of synthetic peptides that normally lead to T cell responses. This condition is reminiscent of the T cell inhibition observed when antagonistic and stimulatory peptides are added to APC at the same time. Our results thus reveal a novel type of interference by pathogens with antigen presentation and T cell stimulation that could give the pathogen a decisive advantage in dissemination and disease.